U.S. patent application number 17/355962 was filed with the patent office on 2021-11-04 for cmp-dependent sialidase activity.
The applicant listed for this patent is Roche Diagnostics Operations, Inc.. Invention is credited to Michael GREIF, Sebastian MALIK, Harald SOBEK, Marco THOMANN.
Application Number | 20210340585 17/355962 |
Document ID | / |
Family ID | 1000005725356 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210340585 |
Kind Code |
A1 |
SOBEK; Harald ; et
al. |
November 4, 2021 |
CMP-DEPENDENT SIALIDASE ACTIVITY
Abstract
The present disclosure is directed to the properties of certain
glycosyltransferase variants having N-terminal truncation deletions
or internal deletions. Any of the mutants disclosed in here exhibit
.alpha.-2,6-sialyltransferase enzymatic activity in the presence of
CMP-activated sialic acid as co-substrate, and in the presence of a
suitable acceptor site. A fundamental finding documented in the
present disclosure is that such enzyme are not only capable of
catalyzing transfer of a sialidyl moiety but they are also capable
of catalyzing hydrolytic cleavage of terminally bound sialic acid
from a glycan.
Inventors: |
SOBEK; Harald; (Biberach,
DE) ; GREIF; Michael; (Penzberg, DE) ;
THOMANN; Marco; (Penzberg, DE) ; MALIK;
Sebastian; (Antdorf, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Diagnostics Operations, Inc. |
Indianapolis |
IN |
US |
|
|
Family ID: |
1000005725356 |
Appl. No.: |
17/355962 |
Filed: |
June 23, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15629040 |
Jun 21, 2017 |
11078511 |
|
|
17355962 |
|
|
|
|
PCT/EP2015/080741 |
Dec 21, 2015 |
|
|
|
15629040 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/1081 20130101;
C12Y 301/03 20130101; C12Y 204/99001 20130101; C07K 2317/41
20130101; C12P 21/005 20130101; C07K 16/00 20130101; C12P 19/44
20130101 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C12N 9/10 20060101 C12N009/10; C12P 19/44 20060101
C12P019/44; C07K 16/00 20060101 C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2014 |
EP |
14199648.8 |
Claims
1-10. (canceled)
11. A method of producing in vitro a sialylated target molecule,
the method comprising the steps of a) providing an aqueous
composition according to claim 1; (b) forming one or more terminal
antennal
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-g-
lucosamine residue(s) by incubating the aqueous composition of step
(a), thereby reacting cytidine-5'-monophospho-N-acetylneuraminic
acid as co-substrate, thereby forming 5'-cytidine-monophosphate;
and (c) hydrolyzing the phosphoester bond of the
5'-cytidine-monophosphate formed in step (b), thereby reducing
5'-cytidine-monophosphate-mediated inhibition, thereby maintaining
the activity of the soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I; thereby
producing in vitro the sialylated target molecule.
12. The method according to claim 11, wherein the method is
performed at a temperature of 0.degree. C. to 40.degree. C.
13. The method according to claim 11, wherein steps (b) and (c) are
performed in the same vessel.
14. The method according to claim 11, wherein steps (b) and (c) are
performed for a period selected from the group consisting of 2 h to
96 h, 2 h to 23 h, 2 h to 6 h, and about 2 h.
15. The method according to claim 11, wherein steps (b) and (c) are
performed for a period selected from the group consisting of 6 h to
96 h, 6 h to 23 h, and about 6 h.
16. The method according to claim 11, wherein steps (b) and (c) are
performed for a period selected from the group consisting of 23 h
to 96 h, and about 23 h.
17. The method according to claim 11, wherein steps (b) and (c) are
performed for a period of about 96 h.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 15/629,040 (published as U.S. Publication No.
2017/0298405) filed Jun. 21, 2017, which is a continuation
application of International Application No. PCT/EP2015/080741
filed Dec. 21, 2015, claiming priority to European Application No.
14199648.8 filed Dec. 22, 2014, the disclosures of which are hereby
incorporated by reference in their entirety.
BACKGROUND
[0002] The present disclosure is directed to the properties of
certain glycosyltransferase variants having N-terminal truncation
deletions or internal deletions. Any of the mutants disclosed in
here exhibit .alpha.-2,6-sialyltransferase enzymatic activity in
the presence of CMP-activated sialic acid as co-substrate, and in
the presence of a suitable acceptor site. A fundamental finding
documented in the present disclosure is that such enzymes are not
only capable of catalyzing transfer of a sialidyl moiety but they
are also capable of catalyzing hydrolytic cleavage of terminally
bound sialic acid from a glycan. Particularly it was found that in
the presence of cytidine-5'-monophosphate (CMP) glycosyltransferase
activity is inhibited, and sialidase activity is stimulated.
Sialidase activity was found to be dependent on the presence of a
particular stretch of amino acids (position 90 to 108) in the
polypeptide sequence of the reference (wildtype) hST6Gal-I
polypeptide. Deletion of this sequence portion in an N-terminal
truncation variant was found to abolish sialidase activity, notably
both in the presence and in the absence of CMP. Thus, disclosed are
compositions, uses and methods employing the CMP-mediated feed-back
regulation documented herein.
[0003] Contrary to previous findings .alpha.-2,6-sialyltransferase
mutants were found to exhibit sialidase enzymatic activity,
particularly (but not limited to) a variant of human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I (hST6Gal-I;
wildtype amino acid sequence see SEQ ID NO:1) with a truncation
deletion involving the first 89 N-terminal amino acids of the
respective wild-type polypeptide (i.e. a mutant with the amino acid
sequence of SEQ ID NO:2). A fundamental finding documented in the
present disclosure is that this mutant enzyme is not only capable
of catalyzing transfer of a sialidyl moiety; in fact, the
.alpha.-2,6-sialyltransferase variant is also capable of catalyzing
hydrolytic cleavage of terminally bound sialic acid from a glycan.
The present disclosure further reports the unexpected observation
of feed-back inhibition. Particularly it was found that in the
presence of cytidine-5'-monophosphate (CMP) glycosyltransferase
activity is inhibited, and sialidase activity is stimulated. Even
more surprising, not only the deletion mutant involving the first
89 N-terminal amino acids but also other N-terminal truncation
variants of human .beta.-galactoside-.alpha.-2,6-sialyltransferase
I (hST6Gal-I) were found to exhibit sialidase enzymatic activity.
However, sialidase activity was found to be dependent on the
presence of a particular stretch of amino acids (position 90 to
108, see FIG. 1) in the polypeptide sequence of the reference
(wildtype) hST6Gal-I polypeptide according to SEQ ID NO:1. Deletion
of this sequence portion in an N-terminal truncation variant was
found to abolish sialidase activity, notably both in the presence
and in the absence of CMP. Thus, disclosed are compositions, uses
and methods employing the CMP-mediated feed-back regulation
documented herein, particularly directed to controlled hydrolysis
of the .alpha.2,6 glycosidic bond in a
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-g-
lucosamine moiety. Further, compositions, uses and methods with a
CMP-insensitive hST6Gal-I are disclosed.
[0004] Transferases (EC 2) catalyze transfer of a functional group
from one substance to another. Glycosyltransferases, a superfamily
of enzymes, are involved in synthesizing the carbohydrate portions
of glycoproteins, glycolipids and glycosaminoglycans. Specific
glycosyltransferases synthesize oligosaccharides by the sequential
transfer of the monosaccharide moiety of an activated sugar donor
to an acceptor molecule. Hence, a "glycosyltransferase" catalyzes
the transfer of a sugar moiety from its nucleotide donor to an
acceptor moiety of a polypeptide, lipid, glycoprotein or
glycolipid. This process is also known as "glycosylation". A
carbohydrate portion which is structural part of e.g. a
glycoprotein is also referred to as "glycan". Glycans constitute
the most prevalent of all known post-translational protein
modifications. Glycans are involved in a wide array of biological
recognition processes as diverse as adhesion, immune response,
neural cell migration and axonal extension. As structural part of
glycoproteins glycans also have a role in protein folding and the
support of protein stability and biological activity.
[0005] In glycosyltransferase catalysis, the monosaccharide units
glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc),
N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA),
galacturonic acid (GalUA) and xylose are activated as uridine
diphosphate (UDP)-.alpha.-D derivatives; arabinose is activated as
a UDP-.beta.-L derivative; mannose (Man) and fucose are activated
as GDP-.alpha.-D and GDP-.beta.-L derivatives, respectively; and
sialic acid (=.beta.-D-Neu5Ac; =Neu5Ac; =SA; =NANA) is activated as
a CMP derivative of sialic acid. CMP-activated sialic acid
(=CMP-.beta.-D-Neu5Ac, see below) appears to be the only naturally
occurring nucleotide sugar in the form of a nucleotide
monophosphate.
[0006] Many different glycosyltransferases contribute to the
synthesis of glycans. The structural diversity of carbohydrate
portions of glycoproteins is particularly large and is determined
by complex biosynthetic pathways. In eukaryotes the
post-translational biosynthesis of the glycan-part of glycoproteins
takes place in the lumen of the endoplasmatic reticulum ("ER") and
the Golgi apparatus. A single (branched or linear) carbohydrate
chain of a glycoprotein is typically a N- or an O-linked glycan.
During post-translational processing, carbohydrates are typically
connected to the polypeptide via asparagine ("N-linked
glycosylation"), or via serine or threonine ("O-linked
glycosylation"). Synthesis of a glycan, no matter whether N- or
O-linked (="N-/O-linked") is effected by the activity of several
different membrane-anchored glycosyltransferases. A glycoprotein
may comprise one or more glycan-connected amino acids
(="glycosylation sites"). A specific glycan structure may be linear
or branched. Branching is a notable feature of carbohydrates which
is in contrast to the linear nature typical for DNA, RNA, and
polypeptides. Combined with the large heterogeneity of their basic
building blocks, the monosaccharides, glycan structures exhibit
high diversity. Furthermore, in members of a particular
glycoprotein species the structure of a glycan attached to a
particular glycosylation site may vary, thus resulting in
microheterogeneity of the respective glycoprotein species, i.e. in
a species sharing the same amino acid sequence of the polypeptide
portion.
[0007] A sialyltransferase (="ST") is a glycosyltransferase that
catalyzes transfer of a sialic acid residue from a donor compound
to (i) a terminal monosaccharide acceptor group of a glycolipid or
a ganglioside, or (ii) to a terminal monosaccharide acceptor group
of an N-/O-linked glycan of a glycoprotein. For the purpose of the
present disclosure, the donor compound is also referred to as
"co-substrate". For mammalian sialyltransferases including human ST
species there is a common donor compound which is
cytidine-5'-monophospho-N-acetylneuraminic acid
(=CMP-.beta.-D-Neu5Ac=CMP-Neu5Ac=CMP-NANA; =CMP-sialic acid;
=CMP-SA). Well known to the skilled person, CMP-sialic acid is a
specific embodiment of a donor compound for a sialyltransferase;
further, there exist functional equivalents including but not
limited to CMP-9-fluoresceinyl-sialic acid. Transfer and covalent
coupling of a sialic acid residue (or the functional equivalent
thereof) to a receptor site is also referred to as "sialylating"
and "sialylation".
[0008] In the glycan structure of a sialylated glycoprotein the
(one or more) sialyl moiety (moieties) is (are) usually found in
the terminal position of an oligosaccharide. Thus, depending on the
amount of sialylated sites, one or more sialic acid residue(s) can
form part of a glycan moiety of a given glycoprotein. Owing to the
terminal, i.e. exposed position, sialic acid can participate in
many different biological recognition phenomena and serve in
different kinds of biological interactions. In a glycoprotein more
than one sialylation site may be present, i.e. a site capable of
serving as a substrate for a sialyltransferase and being an
acceptor group suitable for the transfer of a sialic acid residue.
Such more than one site can in principle be the termini of a
plurality of linear glycan portions anchored at different
glycosylation sites of the glycoprotein. Additionally, a branched
glycan may have a plurality of sites where sialylation can
occur.
[0009] According to current knowledge, a terminal sialic acid
residue can be found (i) .alpha.2.fwdarw.3 (.alpha.2,3) linked to
galactosyl-R, (ii) .alpha.2.fwdarw.6 (.alpha.2,6) linked to
galactosyl-R, (iii) .alpha.2.fwdarw.6 (.alpha.2,6) linked to
N-acetylgalactosaminidyl-R, (iv) .alpha.2.fwdarw.6 (.alpha.2,6)
linked to N-acetylglucosaminidyl-R, and (v) .alpha.2.fwdarw.8/9
(.alpha.2,8/9) linked to sialidyl-R, wherein --R denotes the rest
of the acceptor substrate moiety. Hence, a sialyltransferase active
in the biosynthesis of sialylconjugates is generally named and
classified according to its respective monosaccharide acceptor
substrate and according to the 3, 6 or 8/9 position of the
glycosidic bond it catalyzes. Accordingly, in the literature known
to the art, e.g. in Patel R Y, et al, Glycobiology 16 (2006)
108-116, reference to eukaryotic sialyltransferases is made such as
(i) ST3Gal, (ii) ST6Gal, (iii) ST6GalNAc, or (v) ST8Sia, depending
on the hydroxyl position of the acceptor sugar residue to which the
Neu5Ac residue is transferred while forming a glycosidic bond.
Reference to sialyltransferases in a more generic way can also be
made e.g. as ST3, ST6, ST8; thus, "ST6" specifically encompasses
the sialyltransferases catalyzing an .alpha.2,6 sialylation.
[0010] The disaccharide moiety
.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-glucosamine
(=Gal.beta.1,4GlcNAc) is a frequent terminal residue of the
antennae of N-linked glycans of glycoproteins, but may be also
present in O-linked glycans and in glycolipids. In addition, a
terminal Gal.beta.1,4GlcNAc moiety can be generated in certain
target glycoproteins as a result of galactosyltransferase enzymatic
activity. The enzyme
.beta.-galactoside-.alpha.2,6-sialyltransferase (="ST6Gal") is able
to catalyze .alpha.2,6-sialylation of a terminal Gal.beta.1,4GlcNAc
acceptor moiety of a glycan or a branch of a glycan, also known to
the art as "antenna". For general aspects thereof, reference is
made to the document of DallOlio F. Glycoconjugate Journal 17
(2000) 669-676. In human and in other mammals there appear to be
several species (isozymes) of ST6Gal. The present disclosure
particularly discloses human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I (=hST6Gal-I; EC
2.4.99.1 according to IUBMB Enzyme Nomenclature) and variants
thereof, but is not limited thereto.
[0011] The ST6 group of sialyltransferases comprises two subgroups,
ST6Gal and ST6GalNAc. The activity of ST6Gal enzymes catalyzes
transfer of a Neu5Ac residue to the C6 hydroxyl group of a free
galactosyl residue being part of terminal Gal.beta.1,4GlcNAc in a
glycan or an antenna of a glycan, thereby forming in the glycan a
terminal sialic acid residue .alpha.2.fwdarw.6 linked to the
galactosyl residue of the Gal.beta.1,4GlcNAc moiety. The resulting
newly formed terminal moiety in the glycan is
Neu5Ac.alpha.2,6Gal.beta.1,4GlcNAc.
[0012] The wild-type polypeptide of human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I (hST6Gal-I) at
the time of filing of the present document was disclosed as
"UniProtKB/Swiss-Prot: P15907.1" in the publically accessible NCBI
database (http://www.ncbi.nlm.nih.gov/protein/115445). Further
information including coding sequences are provided as hyperlinks
compiled within the database entry "Gene ID: 6480"
(http://www.ncbi.nlm.nih.gov/gene/6480).
[0013] Mammalian sialyltransferases share with other mammalian
Golgi-resident glycosyltransferases a so-called "type II
architecture" with (i) a short cytoplasmic N-terminal tail, (ii) a
transmembrane fragment followed by (iii) a stem region of variable
length and (iv) a C-terminal catalytic domain facing the lumen of
the Golgi apparatus (Donadio S. et al. in Biochimie 85 (2003)
311-321). Accordingly, a "soluble" sialyltransferase with an
N-terminal truncation deletion lacks at least the elements (i) and
(ii) of the type II architecture. Mammalian sialyltransferases
appear to display significant sequence homology in their catalytic
domain. Recent data regarding structure and function of hST6Gal-I
are disclosed in Kuhn B. et al. (Biol. Crystallography D69 (2013)
1826-1838).
[0014] In certain mammals including mouse, rat and humans, ST6Gal
has widespread tissue distribution. It is particularly abundant in
liver, the major site of serum glycoprotein synthesis (Weinstein J.
et al. J. Biol. Chem 257 (1982) 13835-13844). On the one hand
sialyltransferase exists predominantly in a membrane-bound form
within the Golgi and trans-Golgi network where it participates in
the posttranslational modification of newly synthesized secretory
or cell surface glycoproteins. On the other hand, a soluble form of
ST6Gal-I exists in the serum (Kim Y. S. et al Biochim. Biophys Acta
244 (1971) 505-512; Dalziel M. et al. Glycobiology 9 (1999)
1003-1008) and predominantly is derived from the liver (Kaplan, H.
A. et al. J. Biol. Chern. 258 (1983) 11505-11509; van Dijk, W. et
al. Biochem. Cell. Biol. 64 (1986) 79-84; Dalziel M. et al.
(supra)) by a proteolytic event that liberates the catalytic domain
from its membrane anchor (Kaplan et al., supra; Weinstein, J. et
al. J. Biol. Chem. 262 (1987) 17735-17743). Thus, any variant of an
originally membrane-anchored glycosyltransferase with an N-terminal
truncation comprising the membrane anchor is encompassed by the
term "soluble variant". Soluble variants can exemplarily be
generated by proteolytically removing the portion with the membrane
anchor from the protein, or by expressing a variant nucleic acid
sequence encoding a N-terminally truncated form of the original
protein wherein the truncation includes the membrane anchor
(transmembrane fragment, element (ii) of the type II
architecture).
[0015] Donadio S. et al. (supra) recombinantly expressed several
N-terminally truncated variants of hST6Gal-I without the membrane
anchor in CHO cells. The authors found that N-terminal deletions
comprising the first 35, 48, 60 and 89 amino acids yielded variants
of hST6Gal-I which were enzymatically active and capable of
transferring sialic acid to exogenous acceptors.
[0016] Glycosylation is an important posttranslational modification
of proteins influencing protein folding, stability and regulation
of the biological activity. The sialyl residue is usually exposed
at the terminal position of an N-glycan and therefore, a major
contributor to biological recognition and ligand function. As an
important example, IgG with glycans featuring terminal sialic acid
residues were found to induce reduced inflammatory response and
showed an increase in serum half-life. Therefore, use of
glycosyltransferases for enzymatic synthesis of defined glycan
structures is becoming an engineering tool towards direct in vitro
N-glycosylation of therapeutic proteins, and particularly
therapeutic monoclonal antibodies.
[0017] Since glycosyltransferases of prokaryotic origin usually do
not act on complex glycoprotein structures, sialyltransferases of
mammalian origin are preferred for in vitro glycoengineering
purposes. For example, Barb et al. (2009) prepared highly
sialylated forms of the Fc fragment of immunoglobulin G using
isolated human ST6Gal-I. However, the access to recombinant
hST6Gal-I for such applications is still limited due to low
expression yield and/or poor activity of hST6Gal-I recombinantly
expressed in various hosts (methylotrophic yeast Pichia pastoris,
cultured Spodoptera frugiperda cells, E. coli-based expression
systems).
[0018] Kleineidam R. G. et al. Glycoconjugate Journal (1997) 14:
57-66 disclose a number of inhibitors of
.alpha.-2,6-sialyltransferase from rat liver. Specifically, 70%,
40%, 39% and 71% inhibition of .alpha.-2,6-sialyltransferase was
observed in the presence of Cytidine, 2'-CMP, 3'-CMP and 5'-CMP,
respectively, wherein each inhibitor was tested at a concentration
of 0.25 mM.
[0019] While the use of mammalian glycosyltransferases for in vitro
sialylating a glycosylated target molecule such as a glycoprotein
or a glycolipid is known to the art, the opposite reaction
(sialidase activity, hydrolytic cleavage of a terminal sialyl
residue from a glycan moiety) is typically provided by a
neuraminidase, so far. The original finding by the present
inventors is, however, that a soluble variant of a
sialyltransferase of mammalian origin displays sialidase activity
in the presence of CMP. In fact, the specific example of a soluble
variant of human .beta.-galactoside-.alpha.-2,6-sialyltransferase I
lacking the transmembrane domain by means of a N-terminal
truncation can be used for both, (i) sialylation of a target
glycoprotein and (ii) hydrolytic cleavage of sialyl residues from a
sialylated target glycoprotein. Depending on the presence of CMP
and the interaction of CMP with the soluble variant, sialylation
can be controlled quantitatively. In specific embodiments involving
target molecules with two or more antennal glycan acceptor sites,
the present disclosure provides means, methods and conditions
allowing to sialylate just one out of the several acceptor sites,
as well as sialylating two or more, or even all acceptor sites of
the target molecule.
[0020] This paves the way for a number of different approaches,
particularly in the field of in vitro glycoengineering of
immunoglobulins, and also of other glycosylated target
molecules.
SUMMARY
[0021] In a first aspect and a specific embodiment of all other
aspects as disclosed herein there is disclosed an aqueous
composition comprising [0022] (a) a soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I comprising the
amino acid motif from position 90 to position 108 in SEQ ID NO:1;
[0023] (b) cytidine-5'-monophospho-N-acetylneuraminic acid, or a
functional equivalent thereof; [0024] (c) a glycosylated target
molecule selected from a glycoprotein and a glycolipid, the target
molecule comprising one or more antenna(e), at least one antenna
having as a terminal structure a
.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-glucosamine moiety with a
hydroxyl group at the C6 position in the galactosyl residue; [0025]
(d) an aqueous solution permitting glycosyltransferase enzymatic
activity; wherein the aqueous composition further comprises an
enzyme capable of hydrolyzing the phosphoester bond in
5'-cytidine-monophosphate under conditions permitting
glycosyltransferase enzymatic activity.
[0026] In a second aspect and a specific embodiment of all other
aspects as disclosed herein there is disclosed the use of an enzyme
capable of the hydrolyzing phosphoester bond in
5'-cytidine-monophosphate for maintaining sialyltransferase
enzymatic activity and/or inhibiting sialidase enzymatic activity
in a composition according to the first aspect.
[0027] In a third aspect and a specific embodiment of all other
aspects as disclosed herein there is disclosed a method of
producing in vitro a sialylated target molecule, the method
comprising the steps of [0028] (a) providing an aqueous composition
according to any of the claims 1 and 2; [0029] (b) forming one or
more terminal antennal
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-g-
lucosamine residue(s) by incubating the aqueous composition of step
(a), thereby reacting cytidine-5'-monophospho-N-acetylneuraminic
acid, or a functional equivalent thereof, as co-substrate, thereby
forming 5'-cytidine-monophosphate; [0030] (c) hydrolyzing the
phosphoester bond of the 5'-cytidine-monophosphate formed in step
(b), thereby reducing 5'-cytidine-monophosphate-mediated
inhibition, thereby maintaining the activity of the soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I; thereby
producing in vitro the sialylated target molecule.
[0031] In a fourth aspect and a specific embodiment of all other
aspects as disclosed herein there is disclosed a preparation of
sialylated immunoglobulins, each immunoglobulin having a plurality
of acceptor sites for human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I, wherein less
than about 25% of the acceptor sites in the preparation of
sialylated immunoglobulins are not sialylated, and about 75% or
more are sialylated, wherein the preparation is obtained by a
method according to the third aspect.
[0032] In a fifth aspect and a specific embodiment of all other
aspects as disclosed herein there is disclosed the use of a soluble
human .beta.-galactoside-.alpha.-2,6-sialyltransferase I comprising
the amino acid motif from position 90 to position 108 in SEQ ID
NO:1 for in vitro hydrolyzing in the presence of
5'-cytidine-monophosphate the .alpha.2,6 glycosidic bond in a
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-g-
lucosamine moiety, the moiety being a terminal structure of a
glycan in a sialylated glycoprotein or glycolipid.
DESCRIPTION OF THE FIGURES
[0033] FIG. 1 Representation of the amino acid sequence of the
wild-type hST6Gal-I polypeptide (SEQ ID NO:1), and the N-terminal
portions thereof which are truncated in the deletion variants as
disclosed herein. The deleted positions in the truncations are
symbolized by "X". Underlined are the amino acid at positions
90-108 (SEQ ID NO:1) found to be essential for CMP-induced
Sialidase activity.
[0034] FIG. 2 SDS-PAGE gel after electrophoresis and staining of
the .DELTA.89 hST6Gal-I variant transiently expressed in and
secreted from HEK cells. Lane 1 shows a size-standard, molecular
weights in kDa according to the standard are indicated to the left.
Lane 2: Purified .DELTA.89 hST6Gal-I truncation variant (5 .mu.g of
protein were loaded on the gel).
[0035] FIG. 3 SDS gel after electrophoresis and staining of the
.DELTA.108 hST6Gal-I variant transiently expressed in and secreted
from HEK cells. Lane 1 shows a size-standard, molecular weights in
kDa according to the standard are indicated to the left. Lane 2:
.DELTA.108 hST6Gal-I truncation variant (5 .mu.g of protein were
loaded on the gel).
[0036] FIG. 4 Time course of sialylation of IgG4 MAB using
recombinant .DELTA.89 hST6Gal-I.
[0037] FIG. 5 Kinetics of formation of G2+2SA and G2+1SA, catalyzed
by recombinant .DELTA.89 hST6Gal-I, as shown by mass spectra taken
as a basis for determination of the relative content of the
different sialylated target molecule species.
[0038] FIG. 6 Inhibition of sialidase activity of recombinant
.DELTA.89 hST6Gal-I by CTP. The relative content of antibodies with
glycan with terminal galactose residues (G2+0SA, "asialo"),
mono-sialylated glycan (G2+1SA) and bi-sialylated glycan (G2+2SA)
is shown for different time points.
[0039] FIG. 7 CMP-dependent sialidase activity of .DELTA.89
hST6Gal-I: Incubation of purified IgG1 MAB G2+2SA with .DELTA.89
hST6Gal-I in the absence of CMP. The relative content of antibodies
with glycans with terminal galactose residues (G2+0SA),
monosialylated glycan (G2+1SA) and disialylated glycan (G2+2SA) are
shown for different time points.
[0040] FIG. 8 CMP-dependent sialidase activity of .DELTA.89
hST6Gal-I: Incubation of purified IgG1 MAB G2+2SA with .DELTA.89
hST6Gal-I in the presence of CMP. The relative content of
antibodies with glycans with terminal galactose residues (G2+0SA),
monosialylated glycan (G2+1SA) and disialylated glycan (G2+2SA) are
shown for different time points.
[0041] FIG. 9 CMP-dependent sialidase activity of .DELTA.89
hST6Gal-I: Incubation of purified IgG1 MAB G2+2SA with .DELTA.108
hST6Gal-I in the presence of CMP. The relative content of
antibodies with glycans with terminal galactose residues (G2+0SA),
monosialylated glycan (G2+1SA) and disialylated glycan (G2+2SA) are
shown for different time points.
[0042] FIG. 10 CMP-dependent sialidase activity of .DELTA.89
hST6Gal-I: Incubation of purified IgG1 MAB G2+2SA with
delta57ST3-Gal-I in the presence of CMP. The relative content of
antibodies with glycans with terminal galactose residues (G2+0SA),
monosialylated glycan (G2+1SA) and disialylated glycan (G2+2SA) are
shown for different time points.
[0043] FIG. 11 Sialylation of IgG4 MAB with .DELTA.89 hST6Gal-I in
the absence or presence of 5'-nucleotidase CD73 in the sialylation
reaction mixture. The relative content of antibodies with glycans
with just terminal galactose residues (G2+0SA), monosialylated
glycan (G2+1SA) and disialylated glycan (G2+2SA) is shown. The
amount of 5'-nucleotidase used was 0-0.5 .mu.g. Negative control: 0
.mu.g 5'-nucleotidase CD73.
[0044] FIG. 12 Sialylation of IgG4 MAB with .DELTA.89 hST6Gal-I in
the absence or presence of 5'-nucleotidase CD73 in the sialylation
reaction mixture. The relative content of antibodies with glycans
with just terminal galactose residues (G2+0SA), monosialylated
glycan (G2+1SA) and disialylated glycan (G2+2SA) is shown.
Sialylation reaction mixture with 0.1 .mu.g 5'-nucleotidase
CD73.
[0045] FIG. 13 Sialylation of IgG4 MAB with .DELTA.89 hST6Gal-I in
the absence or presence of 5'-nucleotidase CD73 in the sialylation
reaction mixture. The relative content of antibodies with glycans
with just terminal galactose residues (G2+0SA), monosialylated
glycan (G2+1SA) and disialylated glycan (G2+2SA) is shown.
Sialylation reaction mixture with 0.25 .mu.g 5'-nucleotidase
CD73.
[0046] FIG. 14 Sialylation of IgG4 MAB with .DELTA.89 hST6Gal-I in
the absence or presence of 5'-nucleotidase CD73 in the sialylation
reaction mixture. The relative content of antibodies with glycans
with just terminal galactose residues (G2+0SA), monosialylated
glycan (G2+1SA) and disialylated glycan (G2+2SA) is shown.
Sialylation reaction mixture with 0.5 .mu.g 5'-nucleotidase
CD73.
[0047] FIG. 15 Sialylation of IgG4 MAB with .DELTA.89 hST6Gal-I in
the absence or presence of alkaline phosphatase in the sialylation
reaction mixture. The relative content of antibodies with glycans
with just terminal galactose residues (G2+0SA), monosialylated
glycan (G2+1SA) and disialylated glycan (G2+2SA) is shown. The
amount of 5'-nucleotidase used was 0-100 .mu.g. Negative control: 0
.mu.g alkaline phosphatase.
[0048] FIG. 16 Sialylation of IgG4 MAB with .DELTA.89 hST6Gal-I in
the absence or presence of alkaline phosphatase in the sialylation
reaction mixture. The relative content of antibodies with glycans
with just terminal galactose residues (G2+0SA), monosialylated
glycan (G2+1SA) and disialylated glycan (G2+2SA) is shown.
Sialylation reaction mixture with 1 .mu.g alkaline phosphatase.
[0049] FIG. 17 Sialylation of IgG4 MAB with .DELTA.89 hST6Gal-I in
the absence or presence of alkaline phosphatase in the sialylation
reaction mixture. The relative content of antibodies with glycans
with just terminal galactose residues (G2+0SA), monosialylated
glycan (G2+1SA) and disialylated glycan (G2+2SA) is shown.
Sialylation reaction mixture with 5 .mu.g alkaline phosphatase.
[0050] FIG. 18 Sialylation of IgG4 MAB with .DELTA.89 hST6Gal-I in
the absence or presence of alkaline phosphatase in the sialylation
reaction mixture. The relative content of antibodies with glycans
with just terminal galactose residues (G2+0SA), monosialylated
glycan (G2+1SA) and disialylated glycan (G2+2SA) is shown.
Sialylation reaction mixture with 10 .mu.g alkaline
phosphatase.
[0051] FIG. 19 Sialylation of IgG4 MAB with .DELTA.89 hST6Gal-I in
the absence or presence of alkaline phosphatase in the sialylation
reaction mixture. The relative content of antibodies with glycans
with just terminal galactose residues (G2+0SA), monosialylated
glycan (G2+1SA) and disialylated glycan (G2+2SA) is shown.
Sialylation reaction mixture with 100 .mu.g alkaline
phosphatase.
DETAILED DESCRIPTION
[0052] The terms "a", "an" and "the" generally include plural
referents, i.e. "one or more", unless the context clearly indicates
otherwise. As used herein, "plurality" is understood to mean more
than one. For example, a plurality refers to at least two, three,
four, five, or more. Unless specifically stated or obvious from
context, as used herein, the term "or" is understood to be
inclusive.
[0053] Unless specifically stated or obvious from context, as used
herein, the term "about" is understood as within a range of normal
tolerance in the art, for example within 2 standard deviations of
the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated
value. Unless otherwise clear from context, all numerical values
provided herein can be modified by the term "about".
[0054] The term "amino acid" generally refers to any monomer unit
that can be incorporated into a peptide, polypeptide, or protein.
As used herein, the term "amino acid" includes the following twenty
natural or genetically encoded alpha-amino acids: alanine (Ala or
A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp
or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid
(Glu or E), glycine (Gly or G), histidine (His or H), isoleucine
(Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met
or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or
S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or
Y), and valine (Val or V). In cases where "X" residues are
undefined, these should be defined as "any amino acid." The
structures of these twenty natural amino acids are shown in, e.g.,
Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002).
Additional amino acids, such as selenocysteine and pyrrolysine, can
also be genetically coded for (Stadtman (1996) "Selenocysteine,"
Annu Rev Biochem. 65:83-100 and Ibba et al. (2002) "Genetic code:
introducing pyrrolysine," Curr Biol. 12(13):R464-R466). The term
"amino acid" also includes unnatural amino acids, modified amino
acids (e.g., having modified side chains and/or backbones), and
amino acid analogs. See, e.g., Zhang et al. (2004) "Selective
incorporation of 5-hydroxytryptophan into proteins in mammalian
cells," Proc. Natl. Acad. Sci. U.S.A. 101(24):8882-8887, Anderson
et al. (2004) "An expanded genetic code with a functional
quadruplet codon" Proc. Natl. Acad. Sci. U.S.A. 101(20):7566-7571,
Ikeda et al. (2003) "Synthesis of a novel histidine analogue and
its efficient incorporation into a protein in vivo," Protein Eng.
Des. Sel. 16(9):699-706, Chin et al. (2003) "An Expanded Eukaryotic
Genetic Code," Science 301(5635):964-967, James et al. (2001)
"Kinetic characterization of ribonuclease S mutants containing
photoisomerizable phenylazophenylalanine residues," Protein Eng.
Des. Sel. 14(12):983-991, Kohrer et al. (2001) "Import of amber and
ochre suppressor tRNAs into mammalian cells: A general approach to
site-specific insertion of amino acid analogues into proteins,"
Proc. Natl. Acad. Sci. U.S.A. 98(25):14310-14315, Bacher et al.
(2001) "Selection and Characterization of Escherichia coli Variants
Capable of Growth on an Otherwise Toxic Tryptophan Analogue," J.
Bacteriol. 183(18):5414-5425, Hamano-Takaku et al. (2000) "A Mutant
Escherichia coli Tyrosyl-tRNA Synthetase Utilizes the Unnatural
Amino Acid Azatyrosine More Efficiently than Tyrosine," J. Biol.
Chem. 275(51):40324-40328, and Budisa et al. (2001) "Proteins with
{beta}-(thienopyrrolyl)alanines as alternative chromophores and
pharmaceutically active amino acids," Protein Sci. 10(7):1281-1292.
To further illustrate, an amino acid is typically an organic acid
that includes a substituted or unsubstituted amino group, a
substituted or unsubstituted carboxy group, and one or more side
chains or groups, or analogs of any of these groups. Exemplary side
chains include, e.g., thiol, seleno, sulfonyl, alkyl, aryl, acyl,
keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl,
alkynl, ether, borate, boronate, phospho, phosphono, phosphine,
heterocyclic, enone, imine, aldehyde, ester, thioacid,
hydroxylamine, or any combination of these groups. Other
representative amino acids include, but are not limited to, amino
acids comprising photoactivatable cross-linkers, metal binding
amino acids, spin-labeled amino acids, fluorescent amino acids,
metal-containing amino acids, amino acids with novel functional
groups, amino acids that covalently or noncovalently interact with
other molecules, photocaged and/or photoisomerizable amino acids,
radioactive amino acids, amino acids comprising biotin or a biotin
analog, glycosylated amino acids, other carbohydrate modified amino
acids, amino acids comprising polyethylene glycol or polyether,
heavy atom substituted amino acids, chemically cleavable and/or
photocleavable amino acids, carbon-linked sugar-containing amino
acids, redox-active amino acids, amino thioacid containing amino
acids, and amino acids comprising one or more toxic moieties.
[0055] The term "protein" refers to a polypeptide chain (amino acid
sequence) as a product of the ribosomal translation process,
wherein the polypeptide chain has undergone posttranslational
folding processes resulting in three-dimensional protein structure.
The term "protein" also encompasses polypeptides with one or more
posttranslational modifications such as (but not limited to)
glycosylation, phosphorylation, acetylation and ubiquitination.
[0056] Any protein as disclosed herein, particularly recombinantly
produced protein as disclosed herein, may in a specific embodiment
comprise a "protein tag" which is a peptide sequence genetically
grafted onto the recombinant protein. A protein tag may comprise a
linker sequence with a specific protease cleavage site to
facilitate removal of the tag by proteolysis. As a specific
embodiment, an "affinity tag" is appended to a target protein so
that the target can be purified from its crude biological source
using an affinity technique. For example, the source can be a
transformed host organism expressing the target protein or a
culture supernatant into which the target protein was secreted by
the transformed host organism. Specific embodiments of an affinity
tag include chitin binding protein (CBP), maltose binding protein
(MBP), and glutathione-S-transferase (GST). The poly(His) tag is a
widely-used protein tag which facilitates binding to certain metal
chelating matrices.
[0057] Each of the terms "chimeric protein", "fusion protein" or
"fusion polypeptide" equally refers to a protein whose amino acid
sequence represents a fusion product of subsequences of the amino
acid sequences from at least two distinct proteins. A fusion
protein typically is not produced by direct manipulation of amino
acid sequences, but, rather, is expressed from a "chimeric" gene
that encodes the chimeric amino acid sequence.
[0058] The term "recombinant" refers to an amino acid sequence or a
nucleotide sequence that has been intentionally modified by
recombinant methods. By the term "recombinant nucleic acid" herein
is meant a nucleic acid, originally formed in vitro, in general, by
the manipulation of a nucleic acid by endonucleases, in a form not
normally found in nature. Thus an isolated, mutant DNA polymerase
nucleic acid, in a linear form, or an expression vector formed in
vitro by ligating DNA molecules that are not normally joined, are
both considered recombinant for the purposes of this invention. It
is understood that once a recombinant nucleic acid is made and
reintroduced into a host cell, it will replicate non-recombinantly,
i.e., using the in vivo cellular machinery of the host cell rather
than in vitro manipulations; however, such nucleic acids, once
produced recombinantly, although subsequently replicated
non-recombinantly, are still considered recombinant for the
purposes of the invention. A "recombinant protein" or
"recombinantly produced protein" is a protein made using
recombinant techniques, i.e., through the expression of a
recombinant nucleic acid as depicted above.
[0059] The term "host cell" refers to both single-cellular
prokaryote and eukaryote organisms (e.g., mammalian cells, insect
cells, bacteria, yeast, and actinomycetes) and single cells from
higher order plants or animals when being grown in cell
culture.
[0060] The term "glycosylation" denotes the chemical reaction of
covalently coupling a glycosyl residue to an acceptor group. One
specific acceptor group is a hydroxyl group, e.g. a hydroxyl group
of another sugar. "Sialylation" is a specific form of glycosylation
wherein the acceptor group is reacted with a sialic acid
(=N-acetylneuraminic acid) residue. Such a reaction is typically
catalyzed by a sialyltransferase enzyme using
cytidine-5'-monophospho-N-acetylneuraminic acid as donor compound
or co-substrate.
[0061] "Sialylation" is a specific embodiment of a result of
glycosyltransferase enzymatic activity (sialyltransferase enzymatic
activity in the particular case), under conditions permitting the
same.
[0062] Generally, the skilled person appreciates that the aqueous
composition in which glycosyltransferase enzymatic activity can
take place (=under conditions "permitting glycosyltransferase
enzymatic activity") needs to be buffered using a buffer salt such
as Tris, MES, phosphate, acetate, or another buffer salt
specifically capable of buffering in the pH range of pH 6 to pH 8,
more specifically in the range of pH 6 to pH 7, even more
specifically capable of buffering a solution of about pH 6.5. The
buffer may further contain a neutral salt such as but not limited
to NaCl. Further, in particular embodiments the skilled person may
consider adding to the aqueous buffer a salt comprising a divalent
cation such as Mg.sup.2+ or Mn.sup.2+, e.g., but not limited to,
MgCl.sub.2 and MnCl.sub.2. In additional specific embodiments, the
aqueous composition permitting glycosyltransferase enzymatic
activity may comprise an antioxidant and/or a surfactant.
Conditions permitting glycosyltransferase enzymatic activity known
to the art include ambient (room) temperature, but more generally
temperatures in the range of 0.degree. C. to 40.degree. C.,
particularly 10.degree. C. to 30.degree. C., particularly about
20.degree. C. While the above described conditions provide general
conditions permitting such enzymatic activity, glycosyltransferase
activity further requires the presence of an activated sugar donor
(such as specifically CMP-Neu5Ac) as a co-substrate, in addition.
However, the term "permitting glycosyltransferase enzymatic
activity" is understood as not necessarily including the presence
of the co-substrate. Thus, the term "permitting glycosyltransferase
enzymatic activity" herein also includes conditions permitting the
hydrolysis (sialidase) activity of a mammalian glycosyltransferase
subject of the present disclosure, particularly hydrolysis activity
in the presence of 5'-cytidine-monophosphate (CMP).
[0063] The term "glycan" refers to a poly- or oligosaccharide, i.e.
to a multimeric compound which upon acid hydrolysis yields a
plurality of monosaccharides. A glycoprotein comprises one or more
glycan moieties which are covalently coupled to side groups of the
polypeptide chain, typically via asparagine or arginine ("N-linked
glycosylation") or via serine or threonine ("0-linked
glycosylation").
[0064] The use of glycosyltransferases for enzymatic synthesis of
complex glycan structures is an attractive approach to obtain
complex bioactive glycoproteins. E.g. Barb et al. Biochemistry 48
(2009) 9705-9707 prepared highly potent sialylated forms of the Fc
fragment of immunoglobulin G using isolated human ST6Gal-I.
However, growing interest in the therapeutic application of
glycoproteins leads to an increasing demand of glycosyltransferases
including sialyltransferases. Different strategies to increase or
modify the sialylation of glycoproteins were described by Bork K.
et al. J. Pharm. Sci. 98 (2009) 3499-3508. An attractive strategy
is sialylation in vitro of recombinantly produced proteins (such as
but not limited to immunoglobulins and growth factors),
particularly therapeutic proteins. To this end, several research
groups described expression of sialyltransferases in transformed
organisms and purification of the recombinantly produced
sialyltransferases. As glycosyltransferases of prokaryotic origin
usually do not act on complex glycoproteins (e.g. antibodies),
sialyltransferases from mammalian origin were studied with
preference.
[0065] Particular glycoproteins subject to the disclosures and all
aspects of the present document and the aspects and embodiments
herein comprise without limitation cell surface glycoproteins and
glycoproteins present in soluble form in serum ("serum
glycoprotein"), the glycoproteins particularly being of mammalian
origin. A "cell surface glycoprotein" is understood to be
glycoprotein of which a portion is located on and bound to the
surface of a membrane, by way of a membrane anchor portion of the
surface glycoprotein's polypeptide chain, wherein the membrane is
part of a biological cell. The term cell surface glycoprotein also
encompasses isolated forms of the cell surface glycoprotein as well
as soluble fragments thereof which are separated from the membrane
anchor portion, e.g. by proteolytic cleavage or by recombinant
production of such soluble fragments. A "serum glycoprotein" is
understood as a glycoprotein being present in serum, i.e. a blood
protein present in the non-cellular portion of whole blood, e.g. in
the supernatant following sedimentation of cellular blood
components. Without limitation, a specifically regarded and
embodied serum glycoprotein is an immunoglobulin. Particular
immunoglobulins mentioned in here belong to the IgG group
(characterized by Gamma heavy chains), specifically any of four the
IgG subgroups. For the disclosures, aspects and embodiments herein
the term "serum glycoprotein also encompasses a monoclonal
antibody; monoclonal antibodies artificially are well known to the
art and can be produced e.g. by hybridoma cells or recombinantly
using transformed host cells. A further serum specific glycoprotein
is a carrier protein such as serum albumin, a fetuin, or another
glycoprotein member of the superfamily of histidine-rich
glycoproteins of which the fetuins are members. Further, without
limitation, a specifically regarded and embodied serum glycoprotein
regarding all disclosures, aspects and embodiments herein is a
glycosylated protein signalling molecule. A particular molecule of
this group is erythropoietin (EPO).
[0066] For in vitro engineering of glycoproteins
glycosyltransferases can be used as an efficient tool (Weijers
2008). Glycosyltransferases of mammalian origin are compatible with
glycoproteins as substrates whereas bacterial glycosyltransferases
usually modify simpler substrates like oligosaccharides. For this
reason synthetic changes in the glycan moieties of glycoproteins
are advantageously made using mammalian glycosyltransferases as
tools of choice. However, for a large scale application of
glycosyltransferases in glycoengineering availability of suitable
enzymes in large (i.e. industrial) quantities is required. The
disclosure herein particularly provides proteins with (i) hST6Gal-I
sialyltransferase activity and (ii) sialidase activity which can be
used for quantitatively controlled in vitro sialylation of target
glycoproteins with one or more accessible galactosyl substrate
moiety/moieties.
[0067] Importantly, the amino acid motif in human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I from position 90
to position 108 in SEQ ID NO:1 is required for the enzyme to be
capable of exhibiting sialidase activity. At the same time, this
amino acid motif is required for the enzyme to interact with
5'-CMP. Very remarkably a truncation deletion mutant, a soluble
human .beta.-galactoside-.alpha.-2,6-sialyltransferase I variant
lacking the contiguous N-terminal stretch of the amino acids from
position 1 to position 108 does not exhibit sialidase activity, not
even in the presence of CMP. Thus, it was concluded that the amino
acid motif in human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I from position 90
to position 108 in SEQ ID NO:1 is essential for these properties to
be present.
[0068] Suitable targets to treat with sialidase activity include on
the one hand asialoglycoproteins, i.e. glycoproteins from which
sialic acid residues have been removed by the action of sialidases.
On the other hand, bi-sialylated glycoproteins may serve as
substrate for sialidase activity. Very advantageously, asialo-,
mono-sialylated and bi-sialylated immunoglobulins are specific
substrates, particularly immunoglobulins of the IgG class.
[0069] While expressing wild-type hST6Gal-I in the methylotrophic
yeast Pichia pastoris and having targeted the expressed polypeptide
to the secretory pathway of the host organism, different truncated
variants of recombinantly produced hST6Gal-I were observed.
Generally, hST6Gal-I derived proteins were chromatographically
purified and analyzed, particularly by means of mass spectrometry
and by way of determining the amino acid sequence from the
N-terminus (Edman degradation). By these means truncations,
particularly N-terminal truncations of hST6Gal-I were characterized
in detail.
[0070] Several remarkable truncation variants were identified in
the supernatants of transformed Pichia strains. The variants could
possibly result from site-specific proteolytic cleavage during the
course of secretion from the yeast cells, or result from
endoproteolytic cleavage by one or more extracellular protease(s)
present in the supernatant of cultured Pichia strains.
[0071] Each identified truncation variant was given a "delta"
(=".DELTA.") designation indicating the number of the last amino
acid position of the respective truncation deletion, counted from
the N-Terminus of the wild-type hST6Gal-I polypeptide according to
SEQ ID NO:1 The particular N-terminal truncation variants .DELTA.89
and .DELTA.108 of hST6Gal-I were recombinantly expressed and
studied in more detail.
[0072] Expression vectors were constructed for expression of
hST6Gal-I wild-type protein as well as of the .DELTA.89 and
.DELTA.108 truncation variants in various host organisms including
prokaryotes such as E. coli and Bacillus sp., yeasts such as
Saccharomyces cerevisiae and Pichia pastoris, and mammalian cells
such as CHO cells and HEK cells. Vectors with expression constructs
for the .DELTA.89 and .DELTA.108 truncation variants of hST6Gal-I
were assembled molecularly thereby providing the means of
recombinantly producing the .DELTA.89 variant of human ST6Gal-I in
several transformed host organisms. To facilitate purification of
recombinantly expressed enzymes, the encoded truncation
polypeptides encoded by the constructs optionally included a
N-terminal His-tag, in specific embodiments.
[0073] An aspect and a specific embodiment of all other aspects as
disclosed herein is a variant mammalian glycosyltransferase capable
of catalyzing hydrolysis of the .alpha.2,6 glycosidic bond of a
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-g-
lucosamine moiety of a glycan in a glycoprotein. Particularly, the
variant mammalian glycosyltransferase is capable of catalyzing
formation of the .alpha.2,6 glycosidic bond of a
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-g-
lucosamine moiety in a glycoprotein glycan, thereby generating free
N-acetylneuraminic acid. In a specific embodiment of all aspects as
disclosed herein, the variant mammalian glycosyltransferase is a
soluble enzyme comprising the amino acid motif from position 90 to
position 108 in SEQ ID NO:1 disclosing human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I. Encompassed by
the teachings as disclosed in here are homologous
sialyltransferases comprising an amino acid motif corresponding to
the motif from position 90 to position 108 in SEQ ID NO:1.
[0074] In a specific embodiment of all aspects as disclosed herein,
the variant mammalian glycosyltransferase capable of catalyzing
hydrolysis of the .alpha.2,6 glycosidic bond is a mammalian
glycosyltransferase is derived, by way of amino acid deletion, from
human .beta.-galactoside-.alpha.-2,6-sialyltransferase I according
to SEQ ID NO:1, said sequence being truncated by a deletion from
the N-terminus. In a further specific embodiment of all aspects as
disclosed herein, the truncation deletion from the N-terminus is
the contiguous sequence of position 1 to position 89 of SEQ ID
NO:1.
[0075] Another aspect and a specific embodiment of all other
aspects as disclosed herein is a fusion polypeptide comprising a
polypeptide of a variant mammalian glycosyltransferase according to
any embodiment as disclosed herein. A fusion protein or fusion
polypeptide is a chimeric polypeptide comprising amino acid
sequences of two or more polypeptides. The two or more polypeptides
may have complementary functions, one of the polypeptides may
provide a supplementary functional property, or one of the
polypeptides may have a function unrelated to the others in the
fusion polypeptide. One or more polypeptides comprising organelle
targeting or retention sequences may be fused with a desired
polypeptide to target the desired polypeptide to a specific
cellular organelle, or retain the desired polypeptide within the
cell. One or more polypeptides comprising a carrier sequence that
aids in expression, purification and/or detection of the fusion
polypeptide may be fused with a desired polypeptide (e.g., FLAG, a
myc tag, a 6.times.His tag, GST fusions and the like). Particular
fusion partners include N-terminal leader peptides capable of
directing the variant mammalian glycosyltransferase portion of the
fusion polypeptide to the secretory pathway of the host organism in
which the fusion polypeptide is expressed. Thereby secretion in the
extracellular space and the surrounding medium is facilitated. Yet,
another aspect and a specific embodiment of all other aspects as
disclosed herein is a nucleotide sequence encoding a variant
mammalian glycosyltransferase according to any embodiment as
disclosed herein or a fusion polypeptide comprising as a portion a
variant mammalian glycosyltransferase according to any embodiment
as disclosed herein. Importantly, the nucleotide sequence includes
the sequence from position 90 to position 108 in SEQ ID NO:1, or
the homologous equivalent thereof in the case of a
sialyltransferase homologous to human .beta.-galacto
side-.alpha.-2,6-sialyltransferase I.
[0076] Yet, another aspect and a specific embodiment of all other
aspects as disclosed herein is an expression vector comprising a
target gene and sequences facilitating expression of the target
gene in a host organism transformed with the expression vector,
wherein the target gene comprises a nucleotide sequence as
disclosed herein.
[0077] Yet, another aspect and a specific embodiment of all other
aspects as disclosed herein is a transformed host organism, wherein
the host organism is transformed with an expression vector as
disclosed herein. With particular advantage, Human Embryonic Kidney
293 (HEK) cells can be used to practice the teachings as disclosed
in here. A particular advantage of these cells is that they are
very suited targets for transfection followed by subsequent culture
and transient expression of the target gene. Thus, HEK cells can be
efficiently used to produce target proteins by way of recombinant
expression. With great advantage, expression constructs are
designed to direct the translation products to the secretory
pathway leading to secretion of the variant mammalian
glycosyltransferase or a fusion polypeptide as disclosed herein.
Nevertheless, Jurkat, NIH3T3, HeLa, COS and Chinese Hamster Ovary
(CHO) cells are well-known alternatives and are included herein as
alternative host organisms for transformation and specific
embodiments of all aspects as disclosed herein.
[0078] Yet, another aspect and a specific embodiment of all other
aspects as disclosed herein is a method to produce recombinantly a
variant mammalian glycosyltransferase, the method comprising the
step of expressing in a host organism transformed with an
expression vector a nucleotide sequence encoding a variant
mammalian glycosyltransferase as disclosed herein, wherein a
polypeptide is formed, thereby producing variant mammalian
glycosyltransferase.
[0079] According to earlier knowledge, N-terminally truncated
variants of glycosyltransferases are advantageously used in vitro
due to their lack of transmembrane domains. Thus, such variants are
useful for catalyzing and performing glycosyltransferase reactions
in solution. It was surprisingly found and is disclosed herein that
particularly the N-terminally truncated variant .DELTA.89 hST6Gal-I
displays different activities in vitro, e.g. when incubated with
glycosylated antibodies and in the presence of 5'-CMP. Thus, a
specific embodiment of the present disclosure and all aspects and
embodiments herein is a variant mammalian glycosyltransferase
capable of catalyzing hydrolysis of a .alpha.2,6 glycosidic bond of
a
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-g-
lucosamine moiety of a bi-sialylated glycoprotein, i.e. a
glycoprotein comprising two separate terminal
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-g-
lucosamine moieties in one or more glycan portion(s) of the
glycoprotein. In a specific embodiment, only one .alpha.2,6
glycosidic bond in a is hydrolyzed. In a further specific
embodiment, the bi-sialylated glycoprotein is a bi-sialylated IgG
immunoglobulin.
[0080] As an exemplary case, the IgG-Fc glycan G2 has two galactose
moieties at the termini of the antennate branches which can be
sialylated. Under suitable reaction conditions, the N-terminally
truncated variant .DELTA.89 hST6Gal-I catalyzes the synthesis of
IgG with bi-sialylated G2 glycans (G2+2SA) at the immunoglobulin Fc
portion. However, upon accumulation of 5'-CMP the enzyme variant
acts as a sialidase catalyzing removal by hydrolysis of a sialic
acid moiety from the bi-sialylated (G2+2SA) antibodies resulting in
mono-sialylated (G2+1SA) antibodies. This property was found
unexpectedly and appears to represent an intrinsic sialidase
(neuraminidase) activity.
[0081] In a basic publication on human ST6Gal-I it was stated that
the enzyme does not contain any sialidase activity, see Sticher et
al. Glycoconjugate Journal 8 (1991) 45-54. In view of the present
surprising finding it becomes possible to preferentially synthesize
glycoproteins with mono-, bi- or even higher sialylated glycans,
using the same enzyme and controlling the reaction kinetics of the
enzyme by controlling CMP in the sialylation reaction mixture. A
further advantage is that both activities, sialylation activity and
sialidase activity are provided by the same enzyme, in the same
reaction vessel.
[0082] The general finding documented in the present disclosure is,
however, that there exists a variant mammalian glycosyltransferase,
specifically a glycosyltransferase according to the present
disclosure, which is capable of catalyzing hydrolysis of the
.alpha.2,6 glycosidic bond of a
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl--
.beta.-D-glucosamine moiety of a glycan in a glycoprotein. In
addition to the already known sialyltransferase (sialylation)
activity the surprising finding was that at least as specifically
shown for the N-terminally truncated human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I having the amino
acid sequence of SEQ ID NO:2 there is not only conventional
sialyltransferase but also sialidase enzymatic activity mediated by
this enzyme. Interestingly, in the exemplary cases these two
activities were not observed at the same time, which may partly
explain the unexpected finding. Thus, in the absence of CMP
sialyltransferase activity dominates in the beginning and sialidase
activity becomes apparent only at a later stage during the
incubation, once sufficient amounts of CMP have accumulated.
Nevertheless, the apparent recognition of two distinct activities
of the same enzyme allows to control the extent of sialylation of
target molecules, e.g. by way of varying incubation time.
[0083] However, in a very elegant way, sialylation can be maximized
by adding to the sialylation reaction mixture an enzyme capable of
hydrolyzing the phosphoester bond in 5'-cytidine-monophosphate
under the conditions which permit sialyltransferase enzymatic
activity. Thus, the by-product CMP is removed and the sialylation
catalysis by the sialyltransferase is not counteracted.
[0084] Yet, another aspect and a specific embodiment of all other
aspects as disclosed herein is the use of an enzyme capable of
hydrolyzing the phosphoester bond in 5'-cytidine-monophosphate
under the conditions which permit sialyltransferase enzymatic
activity to maintain glycosyltransferase activity and/or inhibit
sialidase activity of a variant mammalian glycosyltransferase as
disclosed herein, specifically the N-terminally truncated human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I having the amino
acid sequence of SEQ ID NO:2.
[0085] Such controlled sialylation is provided as a novel means to
synthesize in vitro mono-, bi-, and higher sialylated glycoproteins
with a desired degree of sialylation. Thus, though exemplified by
showing the desired technical effects with IgG molecules, the uses
according to the disclosures in here also allow to process other
glycoproteins in a similar way, with the proviso that concerning
glysosyltransferase activity, the glycoproteins comprise two or
more terminal antennate
.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-glucosamine moieties. The
same reasoning applies in an analogous way to glycolipids.
[0086] In a particular example, recombinant humanized IgG1 and IgG4
monoclonal antibodies (mabs), characterized as G2+0SA (=two
acceptor sites present, no sialylation at any acceptor site), as
well as EPO (=erythropoietin) were used as targets in sialylation
experiments (30 .mu.g enzyme/300 .mu.g target protein). .DELTA.89
hST6Gal-I was used under standard reaction conditions and the
G2+0SA, G2+1SA (=mono-sialylation, one out of two acceptor sites is
sialylated) and G2+2SA (=bi-sialylation, both possible acceptor
sites are sialylated) status was analyzed by mass spectrometry.
[0087] Due to the high expression rates and the efficient
purification procedures the exemplary .DELTA.89 hST6Gal-I but also
functionally equivalent enzymes can be made available in large
quantities and with high purity. The variant .DELTA.89 hST6Gal-I
enzyme is active with high molecular weight substrates of which
monoclonal antibodies are just one example. Depending on the
incubation time, .DELTA.89 hST6Gal-I in combination with a
CMP-hydrolyzing enzyme shows good performance in sialylation
experiments using monoclonal antibodies with bi-antennary glycan as
substrate. Using embodiments of the present disclosure the
preferably bi-sialylated glycans are obtained with great advantage
after shorter incubation periods, such as 8 hours. Tetra-antennary
glycans are also accepted as substrate (data not shown). The
results demonstrate technical advantage for in vitro
glycoengineering of therapeutic antibodies.
[0088] The following items further provide specific aspects of the
disclosure, and specific embodiments to practice the teachings
provided herein. [0089] 1. Use of a soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I comprising the
amino acid motif from position 90 to position 108 in SEQ ID NO:1
for in vitro hydrolyzing in the presence of
5'-cytidine-monophosphate the .alpha.2,6 glycosidic bond in a
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-g-
lucosamine moiety, the moiety being a terminal structure of a
glycan in a sialylated glycoprotein or glycolipid. [0090] 2. The
use according to item 1, wherein the soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I comprises the
amino acids from position 90 to position 406 in SEQ ID NO:1. [0091]
3. The use according to any of the items 1 and 2, wherein the amino
acid sequence of the soluble
.beta.-galactoside-.alpha.-2,6-sialyltransferase I is the amino
acid sequence of SEQ ID NO:2. [0092] 4. The use according to any of
the items 1 to 3, wherein the glycoprotein is selected from the
group consisting of a cell surface glycoprotein and a serum
glycoprotein. [0093] 5. The use according to item 4, wherein the
serum glycoprotein is selected from a glycosylated protein
signaling molecule, a glycosylated immunoglobulin, and a
glycosylated protein of viral origin. [0094] 6. The use according
to any of the items 1 to 5, wherein the glycoprotein is
recombinantly produced. [0095] 7. The use according to item 6,
wherein the glycoprotein is recombinantly produced in a transformed
host cell of mammalian origin. [0096] 8. The use according to any
of the items 1 to 7, wherein the glycoprotein is an immunoglobulin
of human origin or a humanized immunoglobulin, the immunoglobulin
being selected from the group consisting of IgG1, IgG2, IgG3, IgG4.
[0097] 9. The use according to any of the items 1 to 7, wherein the
glycoprotein is selected from EPO and asialofetuin. [0098] 10. An
aqueous composition comprising [0099] (a) a soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I comprising the
amino acid motif from position 90 to position 108 in SEQ ID NO:1;
[0100] (b) cytidine-5'-monophospho-N-acetylneuraminic acid, or a
functional equivalent thereof; [0101] (c) a glycosylated target
molecule selected from a glycoprotein and a glycolipid, the target
molecule comprising one or more antenna(e), at least one antenna
having as a terminal structure a
.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-glucosamine moiety with a
hydroxyl group at the C6 position in the galactosyl residue; [0102]
(d) an aqueous solution permitting glycosyltransferase enzymatic
activity; [0103] wherein the aqueous composition further comprises
an enzyme capable of hydrolyzing the phosphoester bond in
5'-cytidine-monophosphate under conditions permitting
glycosyltransferase enzymatic activity. [0104] 11. The aqueous
composition according to item 10, wherein the soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I comprises the
amino acids from position 90 to position 406 in SEQ ID NO:1. [0105]
12. The aqueous composition according to any of the items 10 and
11, wherein the amino acid sequence of the soluble
.beta.-galactoside-.alpha.-2,6-sialyltransferase I is the amino
acid sequence of SEQ ID NO:2. [0106] 13. The aqueous composition
according to any of the items 10 to 12, wherein the glycosylated
target molecule is a glycoprotein selected from the group
consisting of a cell surface glycoprotein and a serum glycoprotein.
[0107] 14. The aqueous composition according to any of the items 10
to 13, wherein the serum glycoprotein is selected from a
glycosylated protein signaling molecule, a glycosylated
immunoglobulin, and a glycosylated protein of viral origin. [0108]
15. The aqueous composition according to any of the items 10 to 14,
wherein the glycoprotein is recombinantly produced. [0109] 16. The
aqueous composition according to item 15, wherein the glycoprotein
is recombinantly produced in a transformed host cell of mammalian
origin. [0110] 17. The aqueous composition according to any of the
items 10 to 16, wherein the glycoprotein is an immunoglobulin of
human origin or a humanized immunoglobulin, the immunoglobulin
being selected from the group consisting of IgG1, IgG2, IgG3, IgG4.
[0111] 18. The aqueous composition according to any of the items 10
to 16, wherein the glycoprotein is selected from EPO and
asialofetuin. [0112] 19. The aqueous composition according to any
of the items 10 to 18, wherein the aqueous solution comprises
water, a buffer salt capable of buffering in the pH range of pH 6
to pH 8, and optionally a compound selected from the group
consisting of a neutral salt, a salt with a divalent cation, an
antioxidant, a surfactant and a mixture thereof. [0113] 20. The
aqueous composition according to any of the items 10 to 19, the
composition having a temperature of 0.degree. C. to 40.degree. C.
[0114] 21. The aqueous composition according to any of the items 10
to 20, wherein the enzyme capable of the hydrolyzing phosphoester
bond in 5'-cytidine-monophosphate is selected from the group
consisting of an alkaline phosphatase, an acid phosphatase, and a
5' nucleotidase. [0115] 22. The aqueous composition according to
item 21, wherein the alkaline phosphatase is selected from the
group consisting of alkaline phosphatase of bacterial origin,
shrimp alkaline phosphatase, calf intestine alkaline phosphatase,
human placental alkaline phosphatase, and a mixture thereof. [0116]
23. The aqueous composition according to item 22, wherein the
aqueous composition further comprises Zn.sup.2+ ions. [0117] 24.
The aqueous composition according to item 21, wherein the 5'
nucleotidase is 5'nucleotidase CD73 of mammalian origin,
specifically of human origin. [0118] 25. Use of an aqueous
composition according to any of the items 10 to 24 for reducing
5'-cytidine-monophosphate-mediated inhibition and thereby
maintaining the sialylating activity of the soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I comprising the
amino acid motif from position 90 to position 108 in SEQ ID NO:1.
[0119] 26. The use according to item 25, wherein the sialylating
activity catalyzes transfer and covalent coupling of the sialic
acid residue, or the functional equivalent thereof, from the
co-substrate to a
.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-glucosamine moiety with a
hydroxyl group at the C6 position in the galactosyl residue, the
moiety being a terminal structure of a glycan of a glycosylated
target molecule selected from a glycoprotein and a glycolipid.
[0120] 27. A method of producing in vitro a sialylated target
molecule, the method comprising the steps of [0121] (a) providing
an aqueous composition according to any of the items 10 to 24;
[0122] (b) forming one or more terminal antennal
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.-
beta.-D-glucosamine residue(s) by incubating the aqueous
composition of step (a), thereby reacting
cytidine-5'-monophospho-N-acetylneuraminic acid, or a functional
equivalent thereof, as co-substrate, thereby forming
5'-cytidine-monophosphate; [0123] (c) hydrolyzing the phosphoester
bond of the 5'-cytidine-monophosphate formed in step (b), thereby
reducing 5'-cytidine-monophosphate-mediated inhibition, thereby
maintaining the activity of the soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I; [0124] thereby
producing in vitro the sialylated target molecule. [0125] 28. The
method according to item 27, wherein the method is performed at a
temperature of 0.degree. C. to 40.degree. C. [0126] 29. The method
according to any of the items 27 and 28, wherein steps (b) and (c)
are performed in the same vessel. [0127] 30. The method according
to any of the items 27 to 29, wherein steps (b) and (c) are
performed for a period selected from the group consisting of 2 h to
96 h, 2 h to 23 h, 2 h to 6 h, and about 2 h. [0128] 31. The method
according to any of the items 27 to 29, wherein steps (b) and (c)
are performed for a period selected from the group consisting of 6
h to 96 h, 6 h to 23 h, and about 6 h. [0129] 32. The method
according to any of the items 27 to 29, wherein steps (b) and (c)
are performed for a period selected from the group consisting of 23
h to 96 h, and about 23 h. [0130] 33. The method according to any
of the items 27 to 29, wherein steps (b) and (c) are performed for
a period of about 96 h. [0131] 34. Use of a soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I lacking the
amino acid motif from position 90 to position 108 in SEQ ID NO:1
for transferring in vitro and in the presence of
5'-cytidine-monophosphate a 5-N-acetylneuraminic acid residue from
the donor compound cytidine-5'-monophospho-N-acetylneuraminic acid,
or from a functional equivalent thereof, to an acceptor, the
acceptor being terminal
.beta.-D-galactosyl-1,4-N-acetyl-3-D-glucosamine in a glycan moiety
of a glycoprotein or a glycolipid. [0132] 35. The use according to
item 34, wherein the soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I comprises the
amino acids from position 109 to position 406 in SEQ ID NO:1.
[0133] 36. The use according to any of the items 34 and 35, wherein
the amino acid sequence of the soluble
.beta.-galactoside-.alpha.-2,6-sialyltransferase I is the amino
acid sequence of SEQ ID NO:5. [0134] 37. The use according to any
of the items 34 to 36, wherein the glycoprotein is selected from
the group consisting of a cell surface glycoprotein and a serum
glycoprotein. [0135] 38. The use according to item 37, wherein the
serum glycoprotein is selected from a glycosylated protein
signaling molecule, a glycosylated immunoglobulin, and a
glycosylated protein of viral origin. [0136] 39. The use according
to any of the items 34 to 38, wherein the glycoprotein is
recombinantly produced. [0137] 40. The use according to item 39,
wherein the glycoprotein is recombinantly produced in a transformed
host cell of mammalian origin. [0138] 41. The use according to any
of the items 34 to 40, wherein the glycoprotein is an
immunoglobulin of human origin or a humanized immunoglobulin, the
immunoglobulin being selected from the group consisting of IgG1,
IgG2, IgG3, IgG4. [0139] 42. The use according to any of the items
34 to 40, wherein the glycoprotein is selected from EPO and
asialofetuin. [0140] 43. An aqueous composition comprising [0141]
(a) a soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I lacking the
amino acid motif from position 90 to position 108 in SEQ ID NO:1;
[0142] (b) cytidine-5'-monophospho-N-acetylneuraminic acid, or a
functional equivalent thereof; [0143] (c) a glycosylated target
molecule selected from a glycoprotein and a glycolipid, the target
molecule comprising one or more antenna(e), at least one antenna
having as a terminal structure a
.beta.-D-galactosyl-1,4-N-acetyl-.beta.-D-glucosamine moiety with a
hydroxyl group at the C6 position in the galactosyl residue; [0144]
(d) an aqueous solution permitting glycosyltransferase enzymatic
activity; [0145] wherein the aqueous composition further comprises
5'-cytidine-monophosphate. [0146] 44. The aqueous composition
according to item 43, wherein the soluble human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I comprises the
amino acids from position 109 to position 406 in SEQ ID NO:1.
[0147] 45. The aqueous composition according to any of the items 43
and 44, wherein the amino acid sequence of the soluble
.beta.-galactoside-.alpha.-2,6-sialyltransferase I is the amino
acid sequence of SEQ ID NO:5. [0148] 46. The aqueous composition
according to any of the items 43 to 45, wherein the glycosylated
target molecule is a glycoprotein selected from the group
consisting of a cell surface glycoprotein and a serum glycoprotein.
[0149] 47. The aqueous composition according to any of the items 43
to 46, wherein the serum glycoprotein is selected from a
glycosylated protein signaling molecule, a glycosylated
immunoglobulin, and a glycosylated protein of viral origin. [0150]
48. The aqueous composition according to any of the items 43 to 47,
wherein the glycoprotein is recombinantly produced. [0151] 49. The
aqueous composition according to item 48, wherein the glycoprotein
is recombinantly produced in a transformed host cell of mammalian
origin. [0152] 50. The aqueous composition according to any of the
items 43 to 49, wherein the glycoprotein is an immunoglobulin of
human origin or a humanized immunoglobulin, the immunoglobulin
being selected from the group consisting of IgG1, IgG2, IgG3, IgG4.
[0153] 51. The aqueous composition according to any of the items 43
to 49, wherein the glycoprotein is selected from EPO and
asialofetuin. [0154] 52. The aqueous composition according to any
of the items 43 to 51, wherein the aqueous solution comprises
water, a buffer salt capable of buffering in the pH range of pH 6
to pH 8, and optionally a compound selected from the group
consisting of a neutral salt, a salt with a divalent cation, an
antioxidant, a surfactant and a mixture thereof. [0155] 53. The
aqueous composition according to any of the items 43 to 52, the
composition having a temperature of 0.degree. C. to 40.degree. C.
[0156] 54. A method of producing in vitro a sialylated target
molecule, the method comprising the steps of [0157] (a) providing
an aqueous composition according to any of the items 43 to 53;
[0158] (b) forming one or more terminal antennal
N-acetylneuraminyl-.alpha.2,6-.beta.-D-galactosyl-1,4-N-acetyl-.-
beta.-D-glucosamine residue(s) by incubating the aqueous
composition of step (a), thereby reacting
cytidine-5'-monophospho-N-acetylneuraminic acid, or a functional
equivalent thereof, as co-substrate, thereby forming
5'-cytidine-monophosphate; [0159] (c) accumulating
5'-cytidine-monophosphate formed in step (b); [0160] thereby
producing in vitro the sialylated target molecule. [0161] 55. The
method according to item 54, wherein the method is performed at a
temperature of 0.degree. C. to 40.degree. C. [0162] 56. The method
according to any of the items 54 and 55, wherein steps (b) and (c)
are performed in the same vessel. [0163] 57. The method according
to any of the items 54 to 56, wherein steps (b) and (c) are
performed for a period selected from the group consisting of 2 h to
72 h. [0164] 58. Use of an enzyme capable of the hydrolyzing
phosphoester bond in 5'-cytidine-monophosphate for maintaining
sialyltransferase enzymatic activity in a composition according to
any of the items 10 to 24. [0165] 59. Use of an enzyme capable of
the hydrolyzing phosphoester bond in 5'-cytidine-monophosphate for
inhibiting sialidase enzymatic activity in a composition according
to any of the items 10 to 24. [0166] 60. A preparation of
sialylated immunoglobulins, each immunoglobulin having a plurality
of acceptor sites for human
.beta.-galactoside-.alpha.-2,6-sialyltransferase I, wherein less
than 25% of the acceptor sites in the preparation of sialylated
immunoglobulins are not sialylated, and 75% or more are sialylated,
wherein the preparation is obtained by a method according to any of
the items 27 to 33.
[0167] 61. The preparation according to item 60, wherein less than
20% of the acceptor sites in the preparation of sialylated
immunoglobulins are not sialylated, and 80% or more are sialylated.
[0168] 62. The preparation according to item 60, wherein less than
10% of the acceptor sites in the preparation of sialylated
immunoglobulins are not sialylated, and 90% or more are
sialylated.
[0169] The Examples that follow are illustrative of specific
embodiments of the disclosure, and various uses thereof. They set
forth for explanatory purposes only, and are not to be taken as
limiting the disclosure.
Example 1
Test for Sialyltransferase Enzymatic Activity
[0170] Asialofetuin (desialylated fetuin, Roche Applied Science)
was used as acceptor and CMP-9-fluoro-NANA
(CMP-9-fluoresceinyl-NeuAc) was used as donor substrate (Brossmer,
R. & Gross H. J. (1994) Meth. Enzymol. 247, 177-193). Enzymatic
activity of a sialyltransferase was determined by measuring the
transfer of sialic acid from the donor compound to asialofetuin.
The reaction mix (35 mM MES, pH 6.0, 0.035% Triton X-100, 0.07%
BSA) contained 2.5 .mu.g of enzyme sample, 5 .mu.L asialofetuin (20
mg/mL) and 2 .mu.L CMP-9-fluoro-NANA (1.0 mg/mL) in a total volume
of 51 .mu.L. The reaction mix was incubated at 37.degree. C. for 30
min. The reaction was stopped by the addition of 10 .mu.L of the
inhibitor CTP (10 mM). The reaction mix was loaded onto a PD10
desalting column equilibrated with 0.1 M Tris/HCl, pH 8.5. Fetuin
was eluted from the column using the equilibration buffer. The
fractions size was 1 mL. The concentration of formed fetuin was
determined using a fluorescence spectrophotometer. Excitation wave
length was 490 nm, emission was measured at 520 nm. Enzymatic
activity was expressed as RFU (relative fluorescence unit). 10,000
RFU/.mu.g is equivalent to a specific activity of 0.0839
nmol/.mu.g.times.min.
Example 2
SDS Gel Electrophoresis
[0171] Analytical SDS gel electrophoresis was carried out using
NuPAGE gels (4-12%, Invitrogen). Samples (36 .mu.L) were diluted
with 12 .mu.L NuPAGE LDS sample buffer (Invitrogen) and incubated
for 2 min at 85.degree. C. Aliquots, typically containing 5 .mu.g
protein were loaded on the gel. The gels were stained using
SimplyBlue SafeStain (Invitrogen).
Example 3
N-Terminal Sequencing by Edman Degradation
[0172] The N-terminal sequences of expressed variants of human
ST6Gal-I were analyzed by Edman degradation using reagents and
devices obtained from Life Technologies. Preparation of the samples
was done as described in the instruction manual of the Life
Technologies ProSorb Sample Preparation cartridges (catalogue
number 401950) and the Life Technologies ProBlott Mini PK/10
membranes (catalogue number 01194). For sequencing the Procise
Protein Sequencing Platform was used.
Example 4
Mass Spectrometry of Glycosylated Human ST6Gal-I Enzymes
[0173] The molecular masses of variants of human ST6Gal-I expressed
in HEK cells were analyzed. Glycosylated forms of human ST6Gal-I
were prepared, and prepared material was analyzed using Micromass
Q-Tof Ultima and Synapt G2 HDMS devices (Waters UK) and MassLynx V
4.1 software.
[0174] For mass spectrometry measurement the samples were buffered
in electrospray medium (20% acetonitrile+1% formic acid). The
buffer exchange was performed with Illustra.TM. MicroSpin.TM. G-25
columns (GE-Healthcare). 20 .mu.g sialyltransferase variant with a
concentration of 1 mg/mL was applied to the pre-equilibrated column
and eluated by centrifugation. The resulting eluate was analyzed by
electrospray ionization mass spectrometry.
Example 5
Mass Spectrometry of Deglycosylated Human ST6Gal-I Enzymes
[0175] The molecular masses of variants of human ST6Gal-I expressed
in HEK cells were analyzed. Delycosylated forms of human ST6Gal-I
were analyzed using Micromass Q-Tof Ultima and Synapt G2 HDMS
devices (Waters UK) and MassLynx V 4.1 software.
[0176] For deglycosylation samples of the sialyltransferase were
denatured and reduced. To 100 .mu.g sialyltransferase 45 .mu.L
denaturing buffer (6 M guanidinium hydrochloride) and 13 .mu.L TCEP
(=tris(2-carboxyethyl)phosphine; 0.1 mM, diluted in denaturing
buffer) were added. Further the appropriate volume of ultrapure
water was added, so that the overall concentration of guanidinium
hydrochloride was about 4 M. After incubation of the sample for 1 h
at 37.degree. C. the buffer was changed using a Bio-SpinR 6 Tris
column (Bio Rad), which was pre-equilibrated with ultrapure water.
The whole sample was applied onto the column and eluted by
centrifugation. To the resulting eluate 5.5 .mu.L of 0.1 U/.mu.L
solution of PNGase-F was added and incubated at 37.degree. C. over
night. Afterwards the samples were adjusted to 30% ACN
(=acetonitrile) and 1% FA (=formamide) and analyzed by electrospray
ionization mass spectrometry.
Example 6
[0177] Cloning of pM1MT Expression Constructs for Transient
Expression in Mammalian Host Cells of Truncated Variant .DELTA.89
of Human ST6Gal-I
[0178] Truncated variant .DELTA.89 of human ST6Gal-I was cloned for
transient expression using an Erythropoietin signal peptide
sequence (Epo) and a peptide spacer of two amino acids ("AP").
[0179] For the Epo-AP-.DELTA.89 hST6Gal-I construct codon-optimized
cDNAs were synthesized, see SEQ ID NO:3. Instead of the natural
leader sequences and the N-terminal protein sequences, the
hST6Gal-I coding region harbors the Erythropoietin signal sequence
plus AP linker sequence in order to ensure correct processing of
expressed polypeptides by the secretion machinery of the host cell
line. In addition, the expression cassettes features SalI and BamHI
restriction sites for cloning into the multiple cloning site of the
predigested pM1MT vector fragment (Roche Applied Science).
Expression of the ST6Gal-I coding sequence is therefore under
control of a human cytomegalovirus (CMV) immediate-early
enhancer/promoter region, followed by an "intron A" for regulated
expression, and a BGH polyadenylation signal.
[0180] Expression of the Epo-AP-.DELTA.89 hST6Gal-I construct in
HEK cells, and secretion of .DELTA.89 hST6Gal-I protein into cell
supernatant was performed as described in Example 8.
Example 7
[0181] Cloning of pM1MT Expression Constructs for Transient
Expression in Mammalian Host Cells of Truncated Variant .DELTA.108
of Human ST6Gal-I
[0182] Truncated variant .DELTA.108 of human ST6Gal-I was cloned
for transient expression using an Erythropoietin signal peptide
sequence (Epo) and a peptide spacer of four amino acids ("APPR").
For the Epo-APPR-.DELTA.108 hST6Gal-I construct a codon-optimized
cDNAs was synthesized, see SEQ ID NO:6. The natural
hST6Gal-I-derived mRNA leader and N-terminal protein sequences were
exchanged with the Erythropoetin signal sequence and the "APPR"
linker sequence to ensure correct processing of the polypeptide by
the secretion machinery of the HEK host cell line. In addition, the
expression cassettes feature SalI and BamHI sites for cloning into
the multiple cloning site of the pre-digested pM1MT vector fragment
(Roche Applied Science). Expression of the hST6Gal-I coding
sequence was thereby put under the control of a human
cytomegalovirus (CMV) immediate-early enhancer/promoter region; the
expression vector further featured an "intron A" for regulated
expression and a BGH polyadenylation signal. Expression of the
Epo-APPR-.DELTA.108 hST6Gal-I construct (SEQ ID NO:6) in HEK cells,
and secretion of .DELTA.108 hST6Gal-I protein into cell supernatant
was performed as described in Example 8.
Example 8
Transformation HEK Cells and Transient Expression and Secretion
[0183] Transient gene expression (TGE) by transfection of plasmid
DNA is a rapid strategy to produce proteins in mammalian cell
culture. For high-level expression of recombinant human proteins a
TGE platform based on a suspension-adapted human embryonic kidney
(HEK) 293 cell line was used. Cells were cultured in shaker flasks
at 37.degree. C. under serum-free medium conditions. The cells were
transfected at approx. 2.times.10.sup.6 vc/mL with the pM1MT
expression plasmids (0.5 to 1 mg/L cell culture) complexed by the
293-Free.TM. (Merck) transfection reagent according to the
manufacturer's guidelines. Three hours post-transfection, valproic
acid, a HDAC inhibitor, was added (final conc. 4 mM) in order to
boost the expression (Backliwal et al. (2008), Nucleic Acids
Research 36, e96). Each day, the culture was supplemented with 6%
(v/v) of a soybean peptone hydrolysate-based feed. The culture
supernatant was collected at day 7 post-transfection by
centrifugation.
Example 9
[0184] Purification of the N-Terminal Truncation Variants of Human
ST6Gal-I from Supernatants of Transformed HEK Cells
[0185] HEK cells were transformed as described in Example 8.
Expression constructs were prepared as described in Examples 6 and
7.
[0186] From supernatants of HEK cell fermentations the two enzyme
variants Epo-AP-.DELTA.89 hST6Gal-I and Epo-APPR-.DELTA.108
hST6Gal-I were purified using a simplified purification protocol.
In a first step, a volume of 0.1 L of culture supernatant was
filtrated (0.2 .mu.m), and the solution was dialysed against buffer
A (20 mM potassium phosphate, pH 6.5). The dialysate was loaded
onto a S-Sepharose.TM. ff (Fast Flow) column (1.6 cm.times.2 cm)
equilibrated with buffer A. After washing with 100 mL buffer A, the
enzyme was eluted with a linear gradient of 10 mL buffer A and 10
mL of buffer A with 200 mM NaCl, followed by a wash step using 48
mL of buffer A with 200 mM NaCl. Fractions (4 mL) were analysed by
an analytical SDS gel electrophoresis.
[0187] Fractions containing the .DELTA.89 hST6Gal-I enzyme were
pooled and dialyzed against buffer B (50 mM MES, pH 6.0). The
dialyzed pool was loaded onto a Heparin Sepharose ff column (0.5
cm.times.5 cm) equilibrated with buffer B and eluted using buffer B
with 200 mM NaCl. Fractions (1 mL) containing the enzyme were
pooled and dialyzed against buffer B. Protein concentrations were
determined at 280 nm (E280 nm [1 mg/mL]=1.931). Mass spectrometry
analysis showed that the recombinantly expressed Epo-AP-.DELTA.89
hST6Gal-I enzyme was secreted without the N-terminal amino acids
AP. This finding was unexpected and indicated unusual cleavage of
the expressed protein by the signal peptidase while removing the
Epo portion. For the recombinant human .DELTA.89 hST6Gal-I enzyme a
specific activity of 3.75 nmol/.mu.g.times.min was determined. FIG.
2 shows the results of a SDS-PAGE of recombinant .DELTA.89
hST6Gal-I variant purified from HEK cells.
[0188] Fractions containing the .DELTA.108 hST6Gal-I enzyme were
pooled and dialyzed against storage buffer (20 mM potassium
phosphate, 100 mM sodium chloride, pH 6.5). Protein concentration
was determined at a wave length of 280 nm using a molar extinction
coefficient of 1.871. Mass spectrometric analysis of the
recombinant protein secreted from the HEK cells transformed with
the Epo-APPR-.DELTA.108-hST6Gal-I expression construct confirmed
the N-terminal sequence "APPR", thus indicating the expected
cleavage of the EPO signal sequence by the signal peptidase. For
the recombinant human .DELTA.108 hST6Gal-I variant from HEK cells a
specific activity of >600 RFU/.mu.g was determined. FIG. 3 shows
the results of a SDS-PAGE of recombinant .DELTA.108 hST6Gal-I
variant purified from HEK cells.
Example 10
[0189] Sialylation of Humanized Monoclonal Antibody IgG4 MAB Using
.DELTA.89 hST6Gal-I
[0190] A highly galactosylated humanized monoclonal antibody IgG4
MAB was used in sialylation experiments. The reaction mixture
contained IgG4 MAB (300 .mu.g in 55 .mu.L 35 mM sodium acetate/Tris
buffer pH 7.0), the donor substrate CMP-NANA (150 .mu.g in 50 .mu.L
water) and sialyltransferase (30 .mu.g .DELTA.89 hST6Gal-I in 20 mM
potassium phosphate, 0.1 M NaCl, pH 6.5). The samples were
incubated at 37.degree. C. for a defined time. To stop the reaction
the samples were frozen at -20.degree. C. For mass analysis 100
.mu.L denaturing buffer (6 M guanidinium chloride) and 30 .mu.L
TCEP (=tris(2-carboxyethyl)phosphine; 0.1 mM, diluted in denaturing
buffer) were added to the samples and the samples were incubated at
37.degree. C. for 1 h. The samples were buffered in electrospray
medium [20% ACN (=acetonitrile), 1% FA (=formamide)] using
pre-equilibrated Illustra.TM. Nap5-Columns (GE-Healthcare). Samples
were analyzed by electrospray ionization mass spectrometry and the
content of G2+0SA, G2+1SA and G2+2SA N-glycans was determined. A
Micromass Q-Tof Ultima and a Synapt G2 HDMS device (Waters UK) were
used, the software used was MassLynx V 4.1. To determine the
kinetics of the sialylation the reaction was incubated up to 72 h.
FIG. 4 shows the relative amounts of differently sialylated target
proteins obtained after different time points during the incubation
period.
[0191] The content of G2+0SA, G2+1SA and G2+2SA was determined by
mass spectrometry. For the variant .DELTA.89 hST6Gal-I already
after 2 h of incubation a high content (88%) of the bi-sialylated
form G2+2SA was obtained, see FIG. 4. The data also show that the
content of G2+0SA and G2+1SA again increased over time due to the
intrinsic CMP-dependent sialidase (neuraminidase) activity of
.DELTA.89 hST6Gal-I. After an incubation of 48 h a G2+1SA content
of 71% was obtained.
[0192] FIG. 5 shows the spectra obtained by mass spectrometric
analysis of different samples of IgG4 MAB. Samples were taken at
time point t=0 (lower panel), time point t=8 h (middle panel) and
time point t=48 h (upper panel). The mass over charge (m/z) signals
of one charge state in the mass spectrum of the IgG molecule with
G2+0SA, G2+1SA and G2+2SA glycans are depicted. The relative
intensities of the different sialylated species are derived from
these signals. Corresponding to FIG. 4, at t=0 h G2+0SA is the
major glycan species. At t=8 h the signal for G2+2SA is the
dominant form whereas at t=48 h, G2+1SA is the most abundant
species. For the determined numerical values see FIG. 4.
Example 11
[0193] Inhibition of Sialidase Activity of .DELTA.89 hST6Gal-I by
CTP
[0194] The compound cytidine-5'-triphosphate (CTP) is a known
potent inhibitor of sialyltransferases (Scudder P R & Chantler
E N BBA 660 (1981) 136-141). To demonstrate that the sialidase
activity is an intrinsic activity of .DELTA.89 hST6Gal-I,
inhibition experiments were performed. In the first phase of the
experiment the sialylation of IgG4 MAB by .DELTA.89 hST6Gal-I was
performed to achieve a high content of G2+2SA (see Example 10).
After 7 h of incubation the G2+2SA content was 94%. Subsequently,
CTP was added to inhibit the sialidase activity of .DELTA.89
hST6Gal-I (final concentration of CTP: 0.67 mM). At different times
samples were taken and the content of G2+0SA, G2+1SA and G2+2SA was
determined by mass spectrometry. The results are shown in FIG. 6.
Compared to inhibitor-free conditions shown in FIG. 4 the
degradation of G2+2SA caused by the sialidase activity was
significantly reduced. After 72 h of incubation 73% of G2+2SA were
still present. The inhibition of the sialidase activity by a known
inhibitor of sialyltransferase activity strongly indicates that
both activities are located in the same active center of .DELTA.89
hST6Gal-I.
Example 12
[0195] Sialylation of IgG1 MAB with .DELTA.89 hST6Gal-I
[0196] The amount of 15 mg of a highly galactosylated humanized
monoclonal antibody IgG1 MAB was used for sialylation treatment.
The reaction mixture contained defined amounts of IgG1 MAB (15 mg
in 1,854 .mu.L aqueous buffer containing 20 mM sodium acetate, 50
mM Tris buffer, pH 8.0), the donor substrate CMP-NANA (7,500 .mu.g
in 2,500 .mu.L water) and sialyltransferase (1,500 .mu.g
recombinantly produced and purified .DELTA.89 hST6Gal-I in 202
.mu.L of 20 mM potassium phosphate, 0.1 M NaCl, pH 6.5). The
components were mixed and the resulting reaction mixture was
incubated at 37.degree. C. for different times, including 2 h, 4 h,
8 h, 24 h, and 48 h. Purification of sialylated antibody was
performed as in Example 13.
[0197] To analyze the degree of sialylation, 124 .mu.L denaturing
buffer (6 M Guanidinium chloride in water) and 30 .mu.L TCEP
(=tris(2-carboxyethyl)phosphine; 0.1 mM, diluted in denaturing
buffer) were added to 76 .mu.L (corresponding to 250 .mu.g IgG1
MAB) and the sample was incubated at 37.degree. C. for 1 h. After
that the sample was buffered in electrospray medium [20% ACN
(=acetonitrile), 1% FA (=formamide)] using pre-equilibrated
Illustra.TM. Nap5-Columns (GE-Healthcare). Subsequently the sample
was analyzed by electrospray ionization mass spectrometry, and the
content of G2+0SA, G2+1SA and G2+2SA N-glycans was determined. A
Synapt G2 HDMS device (Waters UK) was used, the software used was
MassLynx V 4.1.
Example 13
Purification of Sialylated IgG1 MAB
[0198] To remove sialyltransferase and CMP-NANA from the incubated
sialylation reaction mixture of Example 12, incubated IgG1 MAB was
purified using Protein A. The reaction mixture was applied to a
Protein A column equilibrated with 25 mM Tris, 25 mM NaCl, 5 mM
EDTA (=ethylenediaminetetraacetic acid), pH 7. The column was
washed with 25 mM Tris, 25 mM NaCl, 500 mM TMAC
(=tetramethylammonium chloride), 5 mM EDTA pH 5.0 and then with 25
mM Tris, 25 mM NaCl, 5 mM EDTA pH 7.1. IgG1 MAB was eluted with 25
mM Na-Citrate. To avoid spontaneous desilylation at low pH, the pH
was adjusted to pH 7.0 using 1 M Tris pH 9.0. Using this procedure
sialylated IgG1 MAB was obtained in pure form, with a typical yield
of 12 mg.
Example 14
[0199] Sialidase Activity of .DELTA.89 hST6Gal-I on IgG1 MAB in the
Presence and Absence of CMP
[0200] Cytidine monophosphate (5'-CMP, =CMP) is a product of the
reaction catalyzed by sialyltransferase enzymes, generated in the
course of the glycosyltransferase reaction from the co-substrate
CMP-NANA. With incubation time of a sialylation reaction CMP
accumulates in the reaction mixture. To demonstrate that the
inherent sialidase activity is CMP-dependent, highly sialylated
IgG1 monoclonal antibody IgG1 MAB G2+2SA was prepared by incubation
with .DELTA.89 hST6Gal-I in the presence of CMP-NANA, as described
in Example 12, and purified as described in Example 13.
[0201] To an amount of 1,250 .mu.g (in 194 .mu.L) highly sialylated
IgG1 MAB according to Example 12 with an incubation period for
sialylation of 8 h, 125 .mu.g sialyltransferase variant (30
.mu.g/300 .mu.g IgG1 MAB) was added.
[0202] Different N-terminally truncated hST6Gal-I enzyme variants
were tested for CMP-dependent sialidase activity: [0203] .DELTA.89
hST6Gal-I (Example 9) [0204] .DELTA.108 hST6Gal-I (Example 9)
[0205] .DELTA.57 hST3Gal-I (obtained from R&D Systems)
[0206] Four different experiments were made using .DELTA.89
hST6Gal-I (16.8 .mu.L with 125 .mu.g), .DELTA.108 hST6Gal-I (17.3
.mu.L with 125 .mu.g), .DELTA.57 hST3Gal-I (20.1 .mu.L with 125
.mu.g) and a negative control (no enzyme, 20.1 .mu.L ultrapure
water). The enzymes were tested for sialidase activity in the
absence and presence of CMP (10-fold excess based on molarity). The
concentrations were as shown as follows:
.DELTA.89 hST6Gal-I (16.8 .mu.L with 125 .mu.g): 11.8 .mu.g CMP
(c=0.5 mg/mL 23.6 .mu.L) .DELTA.108 hST6Gal-I (17.3 .mu.L with 125
.mu.g): 12.3 .mu.g CMP (c=0.5 mg/mL 24.5 .mu.L) .DELTA.57 hST3Gal-I
(20.1 .mu.L with 125 .mu.g): 12.3 .mu.g CMP (c=0.5 mg/mL 24.5
.mu.L) Negative control (no enzyme): 12.3 .mu.g CMP (c=0.5 mg/mL
24.5 .mu.L)
[0207] The samples were incubated at 37.degree. C. in 20 mM sodium
citrate, 35 mM Tris pH 6.5. Aliquots were taken as samples after
different incubation times, and were analyzed.
[0208] To analyze the degree of sialylation of IgG1 MAB in the
samples, 124 .mu.L denaturing buffer (6 M Guanidinium chloride in
water) and 30 .mu.L TCEP (=tris(2-carboxyethyl)phosphine; 0.1 mM,
diluted in denaturing buffer) were added to 76 .mu.L (corresponding
to 250 .mu.g IgG1 MAB) and the sample was incubated at 37.degree.
C. for 1 h. After that the sample was buffered in electrospray
medium (20% ACN (=acetonitrile), 1% FA (=formamide)) using
pre-equilibrated Illustra.TM. Nap5-Columns (GE-Healthcare).
Subsequently the sample was analyzed by electrospray ionization
mass spectrometry and the content of G2+0SA, G2+1SA and G2+2SA
N-glycans was determined. A Synapt G2 HDMS device (Waters UK) was
used, the software used was MassLynx V 4.1.
[0209] The results are shown in FIGS. 7-10. In the reaction mixture
without CMP no degradation of G2+2SA was observed even after
incubation for 46 h (FIG. 7). Whereas in the presence of CMP a
degradation of G2+2SA was measured accompanied by an increase of
the content of G2+1SA (FIG. 8). Under the conditions described
above .DELTA.89 hST6Gal-I showed a CMP-dependent sialidase
activity, whereas .DELTA.108 hST6Gal-I (FIG. 9) and .DELTA.57
ST3Gal-I (FIG. 10) did not show any sialidase activity in the
presence of CMP. In the latter case this is noted that the enzyme
is specific for 2-3 glycosidic bonds
Example 15
[0210] Sialylation of IgG4 MAB Using .DELTA.89 hST6Gal-I in the
Presence of Phosphatase Enzymatic Activity
[0211] Suppression of CMP-dependent sialidase activity of .DELTA.89
hST6Gal-I was studied by continuous removal of CMP formed during
the reaction.
[0212] In the experiments the enzymes (i) 5'-nucleotidase (EC
3.1.3.5) having a wide specificity for 5'-nucleotides, and (ii)
alkaline phosphatase (EC 3.1.3.1) (both provided by commercial
suppliers) were used. The particular 5'-nucleotidase used here is
also known as ecto-5'-nucleotidase or CD73 (Cluster of
Differentiation 73), in humans encoded by the NT5E gene. Both
enzymes dephosphorylate CMP, i.e. catalyze hydrolysis of the
phosphoester bond in CMP. In the experiments of this Example the
enzymes were used to degrade CMP generated by the sialyltransferase
reaction from the co-substrate CMP-NANA. In the absence of CMP no
intrinsic sialidase activity of .DELTA.89 hST6Gal-I was
observed.
[0213] To 1,000 .mu.g IgG4 MAB (182 .mu.L) 500 .mu.g CMP-NANA (3
mg/mL, 166.7 .mu.L), 100 .mu.g .DELTA.89 hST6Gal-I (13.4 .mu.L, 30
.mu.g/300 .mu.g IgG4 MAB) and different amounts of nucleotidase
(Nu) and alkaline phosphatase (AP) were added. As Zn.sup.2+ ions
are essential for the activity of AP, these were added to a final
concentration of 0.1 mM). The buffer used was 20 mM sodium
acetate/Tris, pH 6.5.
[0214] Different amounts of the enzymes were added to the reaction
mixtures to study the effect of the dephosphorylating enzymes:
[0215] 1) 5'-nucleotidase CD73 was used in a concentration of 0.1
.mu.g/.mu.L. To the reactions 0.10 .mu.g, 0.25 .mu.g and 0.50 .mu.g
were added. [0216] 2) Alkaline phosphatase (AP) was used in a
concentration of 1 .mu.g/.mu.L and 10 .mu.g/.mu.L. To the reactions
1 .mu.g, 5 .mu.g, 10 .mu.g and 100 .mu.g were added.
[0217] After addition of the respective amounts of enzymes the
samples were incubated at 37.degree. C. Samples were taken at
several time points to control the degree of sialylation. Therefore
110 .mu.L denaturing buffer (6 M Guanidinium chloride) and 30 .mu.L
TCEP (0.1 mM, diluted in denaturing buffer) were added to 90 .mu.L
of the sample (about 250 .mu.g IgG4 MAB) and the sample was
incubated at 37.degree. C. for 1 h. After that the sample was
buffered in electrospray medium [20% ACN (=acetonitrile), 1% FA
(=formamide)] using pre-equilibrated Illustra.TM. Nap5-Columns
(GE-Healthcare). Then the sample was analyzed by electrospray
ionization mass spectrometry, and the content of G2+0SA, G2+1SA and
G2+2SA N-glycans was determined. A Synapt G2 HDMS device (Waters
UK) were used, the software used was MassLynx V 4.1.
[0218] Results for sialylation of IgG4 MAB by .DELTA.89 hST6Gal-I
in the absence or presence of 5'-nucleotidase CD73 are depicted in
FIGS. 11-14; and results for sialylation of IgG4 MAB by .DELTA.89
hST6Gal-I in the absence or presence of alkaline phosphatase are
depicted in FIGS. 15-19. As it turned out, introducing a
phosphatase activity capable of hydrolyzing the phosphoester bond
in 5'-CMP effectively reduced CMP-mediated sialidase activity and
promoted sialyltransferase activity.
Sequence CWU 1
1
71406PRTHomo sapiensMISC_FEATUREhST6-Gal-I WT polypeptide 1Met Ile
His Thr Asn Leu Lys Lys Lys Phe Ser Cys Cys Val Leu Val1 5 10 15Phe
Leu Leu Phe Ala Val Ile Cys Val Trp Lys Glu Lys Lys Lys Gly 20 25
30Ser Tyr Tyr Asp Ser Phe Lys Leu Gln Thr Lys Glu Phe Gln Val Leu
35 40 45Lys Ser Leu Gly Lys Leu Ala Met Gly Ser Asp Ser Gln Ser Val
Ser 50 55 60Ser Ser Ser Thr Gln Asp Pro His Arg Gly Arg Gln Thr Leu
Gly Ser65 70 75 80Leu Arg Gly Leu Ala Lys Ala Lys Pro Glu Ala Ser
Phe Gln Val Trp 85 90 95Asn Lys Asp Ser Ser Ser Lys Asn Leu Ile Pro
Arg Leu Gln Lys Ile 100 105 110Trp Lys Asn Tyr Leu Ser Met Asn Lys
Tyr Lys Val Ser Tyr Lys Gly 115 120 125Pro Gly Pro Gly Ile Lys Phe
Ser Ala Glu Ala Leu Arg Cys His Leu 130 135 140Arg Asp His Val Asn
Val Ser Met Val Glu Val Thr Asp Phe Pro Phe145 150 155 160Asn Thr
Ser Glu Trp Glu Gly Tyr Leu Pro Lys Glu Ser Ile Arg Thr 165 170
175Lys Ala Gly Pro Trp Gly Arg Cys Ala Val Val Ser Ser Ala Gly Ser
180 185 190Leu Lys Ser Ser Gln Leu Gly Arg Glu Ile Asp Asp His Asp
Ala Val 195 200 205Leu Arg Phe Asn Gly Ala Pro Thr Ala Asn Phe Gln
Gln Asp Val Gly 210 215 220Thr Lys Thr Thr Ile Arg Leu Met Asn Ser
Gln Leu Val Thr Thr Glu225 230 235 240Lys Arg Phe Leu Lys Asp Ser
Leu Tyr Asn Glu Gly Ile Leu Ile Val 245 250 255Trp Asp Pro Ser Val
Tyr His Ser Asp Ile Pro Lys Trp Tyr Gln Asn 260 265 270Pro Asp Tyr
Asn Phe Phe Asn Asn Tyr Lys Thr Tyr Arg Lys Leu His 275 280 285Pro
Asn Gln Pro Phe Tyr Ile Leu Lys Pro Gln Met Pro Trp Glu Leu 290 295
300Trp Asp Ile Leu Gln Glu Ile Ser Pro Glu Glu Ile Gln Pro Asn
Pro305 310 315 320Pro Ser Ser Gly Met Leu Gly Ile Ile Ile Met Met
Thr Leu Cys Asp 325 330 335Gln Val Asp Ile Tyr Glu Phe Leu Pro Ser
Lys Arg Lys Thr Asp Val 340 345 350Cys Tyr Tyr Tyr Gln Lys Phe Phe
Asp Ser Ala Cys Thr Met Gly Ala 355 360 365Tyr His Pro Leu Leu Tyr
Glu Lys Asn Leu Val Lys His Leu Asn Gln 370 375 380Gly Thr Asp Glu
Asp Ile Tyr Leu Leu Gly Lys Ala Thr Leu Pro Gly385 390 395 400Phe
Arg Thr Ile His Cys 4052317PRTArtificial Sequencedelta89 truncation
variant of hST6Gal-I 2Glu Ala Ser Phe Gln Val Trp Asn Lys Asp Ser
Ser Ser Lys Asn Leu1 5 10 15Ile Pro Arg Leu Gln Lys Ile Trp Lys Asn
Tyr Leu Ser Met Asn Lys 20 25 30Tyr Lys Val Ser Tyr Lys Gly Pro Gly
Pro Gly Ile Lys Phe Ser Ala 35 40 45Glu Ala Leu Arg Cys His Leu Arg
Asp His Val Asn Val Ser Met Val 50 55 60Glu Val Thr Asp Phe Pro Phe
Asn Thr Ser Glu Trp Glu Gly Tyr Leu65 70 75 80Pro Lys Glu Ser Ile
Arg Thr Lys Ala Gly Pro Trp Gly Arg Cys Ala 85 90 95Val Val Ser Ser
Ala Gly Ser Leu Lys Ser Ser Gln Leu Gly Arg Glu 100 105 110Ile Asp
Asp His Asp Ala Val Leu Arg Phe Asn Gly Ala Pro Thr Ala 115 120
125Asn Phe Gln Gln Asp Val Gly Thr Lys Thr Thr Ile Arg Leu Met Asn
130 135 140Ser Gln Leu Val Thr Thr Glu Lys Arg Phe Leu Lys Asp Ser
Leu Tyr145 150 155 160Asn Glu Gly Ile Leu Ile Val Trp Asp Pro Ser
Val Tyr His Ser Asp 165 170 175Ile Pro Lys Trp Tyr Gln Asn Pro Asp
Tyr Asn Phe Phe Asn Asn Tyr 180 185 190Lys Thr Tyr Arg Lys Leu His
Pro Asn Gln Pro Phe Tyr Ile Leu Lys 195 200 205Pro Gln Met Pro Trp
Glu Leu Trp Asp Ile Leu Gln Glu Ile Ser Pro 210 215 220Glu Glu Ile
Gln Pro Asn Pro Pro Ser Ser Gly Met Leu Gly Ile Ile225 230 235
240Ile Met Met Thr Leu Cys Asp Gln Val Asp Ile Tyr Glu Phe Leu Pro
245 250 255Ser Lys Arg Lys Thr Asp Val Cys Tyr Tyr Tyr Gln Lys Phe
Phe Asp 260 265 270Ser Ala Cys Thr Met Gly Ala Tyr His Pro Leu Leu
Tyr Glu Lys Asn 275 280 285Leu Val Lys His Leu Asn Gln Gly Thr Asp
Glu Asp Ile Tyr Leu Leu 290 295 300Gly Lys Ala Thr Leu Pro Gly Phe
Arg Thr Ile His Cys305 310 31531054DNAArtificial SequenceExpression
construct Epo-AP-delta89 ST6 (90-406)misc_recomb(1)..(6)Sal-I
restriction siteCDS(8)..(1048)nucleic acid sequence encoding a
fusion polypeptide of delta89 hST6-Gal-I, N-terminally fused to the
Epo leader peptide and containing an "AP" joining
sequencemisc_feature(95)..(1048)portion ot the nucleic acid
sequence encoding the delta89 hST6-Gal-I portion of the fusion
polypeptidemisc_recomb(1049)..(1054)BamH-I restriction site
3gtcgacc atg ggc gtg cac gaa tgt cct gcc tgg ctg tgg ctg ctg ctg 49
Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu 1 5 10agc
ctg ctg tct ctg cct ctg gga ctg cct gtg ctg ggc gcc cct gaa 97Ser
Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Glu15 20 25
30gcc tct ttc cag gtg tgg aac aag gac agc agc tcc aag aac ctg atc
145Ala Ser Phe Gln Val Trp Asn Lys Asp Ser Ser Ser Lys Asn Leu Ile
35 40 45ccc cgg ctg cag aag atc tgg aag aac tac ctg agc atg aac aag
tac 193Pro Arg Leu Gln Lys Ile Trp Lys Asn Tyr Leu Ser Met Asn Lys
Tyr 50 55 60aag gtg tcc tac aag ggc cct ggc cct ggc atc aag ttt agc
gcc gag 241Lys Val Ser Tyr Lys Gly Pro Gly Pro Gly Ile Lys Phe Ser
Ala Glu 65 70 75gcc ctg aga tgc cac ctg agg gat cac gtg aac gtg tcc
atg gtg gaa 289Ala Leu Arg Cys His Leu Arg Asp His Val Asn Val Ser
Met Val Glu 80 85 90gtg acc gac ttc cca ttc aac acc agc gag tgg gag
ggc tac ctg ccc 337Val Thr Asp Phe Pro Phe Asn Thr Ser Glu Trp Glu
Gly Tyr Leu Pro95 100 105 110aaa gag agc atc cgg acc aaa gcc ggc
cct tgg gga aga tgt gcc gtg 385Lys Glu Ser Ile Arg Thr Lys Ala Gly
Pro Trp Gly Arg Cys Ala Val 115 120 125gtg tct agc gcc ggc agc ctg
aag agt agc cag ctg ggc aga gag atc 433Val Ser Ser Ala Gly Ser Leu
Lys Ser Ser Gln Leu Gly Arg Glu Ile 130 135 140gac gac cac gac gcc
gtg ctg cgg ttc aat ggc gct ccc acc gcc aac 481Asp Asp His Asp Ala
Val Leu Arg Phe Asn Gly Ala Pro Thr Ala Asn 145 150 155ttc cag cag
gac gtg ggc acc aag acc acc atc cgg ctg atg aac tcc 529Phe Gln Gln
Asp Val Gly Thr Lys Thr Thr Ile Arg Leu Met Asn Ser 160 165 170cag
ctc gtg aca acc gag aag cgg ttc ctg aag gac agc ctg tac aac 577Gln
Leu Val Thr Thr Glu Lys Arg Phe Leu Lys Asp Ser Leu Tyr Asn175 180
185 190gag ggc atc ctg atc gtg tgg gac ccc agc gtg tac cac agc gac
atc 625Glu Gly Ile Leu Ile Val Trp Asp Pro Ser Val Tyr His Ser Asp
Ile 195 200 205ccc aag tgg tat cag aac ccc gac tac aac ttc ttc aac
aac tac aag 673Pro Lys Trp Tyr Gln Asn Pro Asp Tyr Asn Phe Phe Asn
Asn Tyr Lys 210 215 220acc tac cgg aag ctg cac ccc aac cag ccc ttc
tac atc ctg aag ccc 721Thr Tyr Arg Lys Leu His Pro Asn Gln Pro Phe
Tyr Ile Leu Lys Pro 225 230 235cag atg ccc tgg gag ctg tgg gac att
ctg cag gaa atc agc ccc gaa 769Gln Met Pro Trp Glu Leu Trp Asp Ile
Leu Gln Glu Ile Ser Pro Glu 240 245 250gag atc cag ccc aac ccc cct
agc tct ggc atg ctg ggc atc att atc 817Glu Ile Gln Pro Asn Pro Pro
Ser Ser Gly Met Leu Gly Ile Ile Ile255 260 265 270atg atg acc ctg
tgc gac cag gtg gac atc tac gag ttt ctg ccc tcc 865Met Met Thr Leu
Cys Asp Gln Val Asp Ile Tyr Glu Phe Leu Pro Ser 275 280 285aag aga
aag acc gac gtg tgc tac tac tac cag aag ttc ttc gac agc 913Lys Arg
Lys Thr Asp Val Cys Tyr Tyr Tyr Gln Lys Phe Phe Asp Ser 290 295
300gcc tgc acc atg gga gcc tac cac cct ctg ctg tac gag aag aac ctc
961Ala Cys Thr Met Gly Ala Tyr His Pro Leu Leu Tyr Glu Lys Asn Leu
305 310 315gtg aag cac ctg aac cag ggc acc gac gag gat atc tac ctg
ctg ggc 1009Val Lys His Leu Asn Gln Gly Thr Asp Glu Asp Ile Tyr Leu
Leu Gly 320 325 330aag gcc acc ctg ccc ggc ttc aga acc atc cac tgc
tga ggatcc 1054Lys Ala Thr Leu Pro Gly Phe Arg Thr Ile His Cys335
340 3454346PRTArtificial SequenceSynthetic Construct 4Met Gly Val
His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu1 5 10 15Leu Ser
Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Glu Ala Ser 20 25 30Phe
Gln Val Trp Asn Lys Asp Ser Ser Ser Lys Asn Leu Ile Pro Arg 35 40
45Leu Gln Lys Ile Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys Val
50 55 60Ser Tyr Lys Gly Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu Ala
Leu65 70 75 80Arg Cys His Leu Arg Asp His Val Asn Val Ser Met Val
Glu Val Thr 85 90 95Asp Phe Pro Phe Asn Thr Ser Glu Trp Glu Gly Tyr
Leu Pro Lys Glu 100 105 110Ser Ile Arg Thr Lys Ala Gly Pro Trp Gly
Arg Cys Ala Val Val Ser 115 120 125Ser Ala Gly Ser Leu Lys Ser Ser
Gln Leu Gly Arg Glu Ile Asp Asp 130 135 140His Asp Ala Val Leu Arg
Phe Asn Gly Ala Pro Thr Ala Asn Phe Gln145 150 155 160Gln Asp Val
Gly Thr Lys Thr Thr Ile Arg Leu Met Asn Ser Gln Leu 165 170 175Val
Thr Thr Glu Lys Arg Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly 180 185
190Ile Leu Ile Val Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys
195 200 205Trp Tyr Gln Asn Pro Asp Tyr Asn Phe Phe Asn Asn Tyr Lys
Thr Tyr 210 215 220Arg Lys Leu His Pro Asn Gln Pro Phe Tyr Ile Leu
Lys Pro Gln Met225 230 235 240Pro Trp Glu Leu Trp Asp Ile Leu Gln
Glu Ile Ser Pro Glu Glu Ile 245 250 255Gln Pro Asn Pro Pro Ser Ser
Gly Met Leu Gly Ile Ile Ile Met Met 260 265 270Thr Leu Cys Asp Gln
Val Asp Ile Tyr Glu Phe Leu Pro Ser Lys Arg 275 280 285Lys Thr Asp
Val Cys Tyr Tyr Tyr Gln Lys Phe Phe Asp Ser Ala Cys 290 295 300Thr
Met Gly Ala Tyr His Pro Leu Leu Tyr Glu Lys Asn Leu Val Lys305 310
315 320His Leu Asn Gln Gly Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys
Ala 325 330 335Thr Leu Pro Gly Phe Arg Thr Ile His Cys 340
3455298PRTArtificial Sequencedelta108 truncation variant of
hST6Gal-I 5Leu Gln Lys Ile Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr
Lys Val1 5 10 15Ser Tyr Lys Gly Pro Gly Pro Gly Ile Lys Phe Ser Ala
Glu Ala Leu 20 25 30Arg Cys His Leu Arg Asp His Val Asn Val Ser Met
Val Glu Val Thr 35 40 45Asp Phe Pro Phe Asn Thr Ser Glu Trp Glu Gly
Tyr Leu Pro Lys Glu 50 55 60Ser Ile Arg Thr Lys Ala Gly Pro Trp Gly
Arg Cys Ala Val Val Ser65 70 75 80Ser Ala Gly Ser Leu Lys Ser Ser
Gln Leu Gly Arg Glu Ile Asp Asp 85 90 95His Asp Ala Val Leu Arg Phe
Asn Gly Ala Pro Thr Ala Asn Phe Gln 100 105 110Gln Asp Val Gly Thr
Lys Thr Thr Ile Arg Leu Met Asn Ser Gln Leu 115 120 125Val Thr Thr
Glu Lys Arg Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly 130 135 140Ile
Leu Ile Val Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys145 150
155 160Trp Tyr Gln Asn Pro Asp Tyr Asn Phe Phe Asn Asn Tyr Lys Thr
Tyr 165 170 175Arg Lys Leu His Pro Asn Gln Pro Phe Tyr Ile Leu Lys
Pro Gln Met 180 185 190Pro Trp Glu Leu Trp Asp Ile Leu Gln Glu Ile
Ser Pro Glu Glu Ile 195 200 205Gln Pro Asn Pro Pro Ser Ser Gly Met
Leu Gly Ile Ile Ile Met Met 210 215 220Thr Leu Cys Asp Gln Val Asp
Ile Tyr Glu Phe Leu Pro Ser Lys Arg225 230 235 240Lys Thr Asp Val
Cys Tyr Tyr Tyr Gln Lys Phe Phe Asp Ser Ala Cys 245 250 255Thr Met
Gly Ala Tyr His Pro Leu Leu Tyr Glu Lys Asn Leu Val Lys 260 265
270His Leu Asn Gln Gly Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys Ala
275 280 285Thr Leu Pro Gly Phe Arg Thr Ile His Cys 290
29561003DNAArtificial SequenceExpression construct Epo-AP-delta108
ST6 (109-406)CDS(8)..(997)open reading frame 6gtcgacc atg ggc gtg
cac gaa tgt cct gcc tgg ctg tgg ctg ctg ctg 49 Met Gly Val His Glu
Cys Pro Ala Trp Leu Trp Leu Leu Leu 1 5 10agc ctg ctg tct ctg cct
ctg gga ctg cct gtg ctg ggc gcc cct cct 97Ser Leu Leu Ser Leu Pro
Leu Gly Leu Pro Val Leu Gly Ala Pro Pro15 20 25 30aga ctg cag aag
atc tgg aag aac tac ctg agc atg aac aag tac aag 145Arg Leu Gln Lys
Ile Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys 35 40 45gtg tcc tac
aag ggc cct ggc cct ggc atc aag ttt agc gcc gag gcc 193Val Ser Tyr
Lys Gly Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu Ala 50 55 60ctg aga
tgc cac ctg agg gat cac gtg aac gtg tcc atg gtg gaa gtg 241Leu Arg
Cys His Leu Arg Asp His Val Asn Val Ser Met Val Glu Val 65 70 75acc
gac ttc cca ttc aac acc agc gag tgg gag ggc tac ctg ccc aaa 289Thr
Asp Phe Pro Phe Asn Thr Ser Glu Trp Glu Gly Tyr Leu Pro Lys 80 85
90gag agc atc cgg acc aaa gcc ggc cct tgg gga aga tgt gcc gtg gtg
337Glu Ser Ile Arg Thr Lys Ala Gly Pro Trp Gly Arg Cys Ala Val
Val95 100 105 110tct agc gcc ggc agc ctg aag agt agc cag ctg ggc
aga gag atc gac 385Ser Ser Ala Gly Ser Leu Lys Ser Ser Gln Leu Gly
Arg Glu Ile Asp 115 120 125gac cac gac gcc gtg ctg cgg ttc aat ggc
gct ccc acc gcc aac ttc 433Asp His Asp Ala Val Leu Arg Phe Asn Gly
Ala Pro Thr Ala Asn Phe 130 135 140cag cag gac gtg ggc acc aag acc
acc atc cgg ctg atg aac tcc cag 481Gln Gln Asp Val Gly Thr Lys Thr
Thr Ile Arg Leu Met Asn Ser Gln 145 150 155ctc gtg aca acc gag aag
cgg ttc ctg aag gac agc ctg tac aac gag 529Leu Val Thr Thr Glu Lys
Arg Phe Leu Lys Asp Ser Leu Tyr Asn Glu 160 165 170ggc atc ctg atc
gtg tgg gac ccc agc gtg tac cac agc gac atc ccc 577Gly Ile Leu Ile
Val Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro175 180 185 190aag
tgg tat cag aac ccc gac tac aac ttc ttc aac aac tac aag acc 625Lys
Trp Tyr Gln Asn Pro Asp Tyr Asn Phe Phe Asn Asn Tyr Lys Thr 195 200
205tac cgg aag ctg cac ccc aac cag ccc ttc tac atc ctg aag ccc cag
673Tyr Arg Lys Leu His Pro Asn Gln Pro Phe Tyr Ile Leu Lys Pro Gln
210 215 220atg ccc tgg gag ctg tgg gac att ctg cag gaa atc agc ccc
gaa gag 721Met Pro Trp Glu Leu Trp Asp Ile Leu Gln Glu Ile Ser Pro
Glu Glu 225 230 235atc cag ccc aac ccc cct agc tct ggc atg ctg ggc
atc att atc atg 769Ile Gln Pro Asn Pro Pro Ser Ser Gly Met Leu Gly
Ile Ile Ile Met 240 245 250atg acc ctg tgc gac cag gtg gac atc tac
gag ttt ctg ccc tcc aag 817Met Thr Leu Cys Asp Gln Val Asp Ile Tyr
Glu Phe Leu Pro Ser Lys255
260 265 270aga aag acc gac gtg tgc tac tac tac cag aag ttc ttc gac
agc gcc 865Arg Lys Thr Asp Val Cys Tyr Tyr Tyr Gln Lys Phe Phe Asp
Ser Ala 275 280 285tgc acc atg gga gcc tac cac cct ctg ctg tac gag
aag aac ctc gtg 913Cys Thr Met Gly Ala Tyr His Pro Leu Leu Tyr Glu
Lys Asn Leu Val 290 295 300aag cac ctg aac cag ggc acc gac gag gat
atc tac ctg ctg ggc aag 961Lys His Leu Asn Gln Gly Thr Asp Glu Asp
Ile Tyr Leu Leu Gly Lys 305 310 315gcc acc ctg ccc ggc ttc aga acc
atc cac tgc tga ggatcc 1003Ala Thr Leu Pro Gly Phe Arg Thr Ile His
Cys 320 3257329PRTArtificial SequenceSynthetic Construct 7Met Gly
Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu1 5 10 15Leu
Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25
30Gln Lys Ile Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys Val Ser
35 40 45Tyr Lys Gly Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu Ala Leu
Arg 50 55 60Cys His Leu Arg Asp His Val Asn Val Ser Met Val Glu Val
Thr Asp65 70 75 80Phe Pro Phe Asn Thr Ser Glu Trp Glu Gly Tyr Leu
Pro Lys Glu Ser 85 90 95Ile Arg Thr Lys Ala Gly Pro Trp Gly Arg Cys
Ala Val Val Ser Ser 100 105 110Ala Gly Ser Leu Lys Ser Ser Gln Leu
Gly Arg Glu Ile Asp Asp His 115 120 125Asp Ala Val Leu Arg Phe Asn
Gly Ala Pro Thr Ala Asn Phe Gln Gln 130 135 140Asp Val Gly Thr Lys
Thr Thr Ile Arg Leu Met Asn Ser Gln Leu Val145 150 155 160Thr Thr
Glu Lys Arg Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly Ile 165 170
175Leu Ile Val Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys Trp
180 185 190Tyr Gln Asn Pro Asp Tyr Asn Phe Phe Asn Asn Tyr Lys Thr
Tyr Arg 195 200 205Lys Leu His Pro Asn Gln Pro Phe Tyr Ile Leu Lys
Pro Gln Met Pro 210 215 220Trp Glu Leu Trp Asp Ile Leu Gln Glu Ile
Ser Pro Glu Glu Ile Gln225 230 235 240Pro Asn Pro Pro Ser Ser Gly
Met Leu Gly Ile Ile Ile Met Met Thr 245 250 255Leu Cys Asp Gln Val
Asp Ile Tyr Glu Phe Leu Pro Ser Lys Arg Lys 260 265 270Thr Asp Val
Cys Tyr Tyr Tyr Gln Lys Phe Phe Asp Ser Ala Cys Thr 275 280 285Met
Gly Ala Tyr His Pro Leu Leu Tyr Glu Lys Asn Leu Val Lys His 290 295
300Leu Asn Gln Gly Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys Ala
Thr305 310 315 320Leu Pro Gly Phe Arg Thr Ile His Cys 325
* * * * *
References